JP2022515566A - Chloride-induced prokaryotic expression system - Google Patents

Abstract

The present invention is directed to the use of recombinant bacteria, recombinant plasmids, pharmaceutical compositions, and kits, as well as reprepared media containing chloride ions for repreparing recombinant bacteria.

Description

The present invention is directed to the use of recombinant bacteria, recombinant plasmids, pharmaceutical compositions, and kits, as well as reprepared media containing chloride ions for repreparing recombinant bacteria.

Biopharmacy or biotherapeutic methods relate to a wide range of medical products produced, for example, by means of biological processes involving recombinant DNA technology.

The biopharmacy or biotherapeutic method is, for example, a polypeptide that is the same as or almost identical to the polypeptide of interest to be treated, such as the hematopoietic stimulating protein erythropoetin, biosynthetic human insulin and its analogs, or specific cell types, specific. Gene expression or translation by neutralizing a monoclonal antibody that can be specifically made to block or block a given target, such as a specific polypeptide, or an endogenous antigen, and the target mRNA molecule. Includes RNA molecules designed to regulate.

Biopharmacy has profound impact on many medical areas of medicine, including some for which no effective treatment was available, and others for which previous treatments were clearly inadequate. Add major treatment options for the treatment of many diseases.

However, because the cost of biotherapy is dramatically higher than traditional medicines, the advent of biopharmacy also raises complex regulatory issues and significant pharmacoeconomic concerns. This factor is especially important because many biopharmaceuticals are used for the rest of their lives, either for the treatment of chronic diseases or for the treatment of cancers that are otherwise incurable.

Moreover, biopharmacy often has the disadvantage of limited stability to degradation by, for example, proteases or nucleases, especially after application to the subject. Partial or complete degradation of the biopharmacy molecule leads to a significantly reduced half-life of the biopharmacy after administration to the subject.

In order to provide a sufficient amount of each biopharmacy to a subject, an alternative mode of application is often required instead of direct application of the biopharmacy to the subject.

For example, biopharmacy such as a polypeptide is transferred by an autologous or heterologous cell secreting each polypeptide, or by using a gene transfer method, eg, a viral vector, to a target tissue for a gene encoding the required polypeptide. It can be provided either by.

A commercially available example is a dermal substitute containing human germ cells, which secretes various growth factors after application to diseased wound areas.

The dermis substitute Dermagraft is composed of fibroblasts, extracellular matrix, and bioabsorbable substrate. Dermagraf is produced from human fibroblasts derived from donor neonatal foreskin tissue. During the manufacturing process, human fibroblasts are seeded on a bioabsorbable polygalactin substrate.

The commercially available dermis substitute Appligraf contains two cell types derived from the neonatal foreskin. Live human keratinocytes and fibroblasts are embedded in the wound type 1 collagen matrix.

The disadvantage of the dermis substitute mentioned earlier is the fairly high price that comes from the manufacturing process. Furthermore, each cell used as a dermal substitute is a human virus, such as immunodeficiency virus types 1 and 2, hepatitis B virus, hepatitis C virus, syphilis, human T lymphocyte tropic type 1 and 2, It must also be tested for evidence of infection with the Epstein-Barr virus.

Another commercially available example is Voretigene neparbobeck-rzyl (LUXTURNA). LUXTURNA is a suspension of gene therapy based on an adeno-associated virus vector for subretinal injections in the retina of individuals with reduced or absent levels of bioactive human retinal pigment epithelium 65 kDa protein (RPE65). It is designed to deliver to cells a normal copy of the gene encoding RPE65.

The disadvantages of gene therapy products based on viral products or any other viral vector are high manufacturing costs and limitations for scaling up production.

For example, WO9714806A2 describes the delivery of a bioactive polypeptide to a subject by a non-invasive bacterium.

WO9611277A1 targets the use of microorganisms as vehicles for the delivery of therapeutic compounds to subjects.

WO2011160062A2 provides a method for treating inflammatory bowel disease and targets recombinant microorganisms capable of in situ producing therapeutically effective amounts of interleukin 27 (IL-27) or variants or fragments thereof in the intestinal mucosa. Including administration to.

US2013209407A is E.I. Targeting commensal strains of coli, which can colonize the urogenital and / or gastrointestinal mucosa, and which have the activity of infectious and / or disease-causing pathogens by secreting heterologous antimicrobial polypeptides. Can block.

Several expression systems for heterologous polypeptides using either inductive or constitutive promoters are known in bacteria.

The constitutive promoter is preferably intracellularly active in all situations and allows preferably high levels of production of the desired heterologous polypeptide, however, it has a significantly increased metabolic load on the bacteria used. And, therefore, reduced viability and / or growth rate can also be reached. This can again have a direct impact on the bacterial yield during the fermentation of the manufacturing process. The higher the metabolic load, the lower the yield during the fermentation process can be.

In contrast, controlled and preferably inducible promoters become active or enhance promoter activity in response to specific inducing factors. Application of the inducing factor to a recombinant bacterium expressing the desired heterologous polypeptide under the control of each controlled and preferably inducible promoter activates the promoter and the nucleic acid of interest encoding each heterologous polypeptide. Is expressed. Preferably, this is an advantage during the production of recombinant bacteria. In that, higher yields of recombinant bacteria can be achieved because the metabolic load is minimized.

A widely used controlled gene expression system is, for example, the Lactococcus lactis nisin-controlled gene expression system (NICE).

Nisin, which is well known to those of skill in the art, includes several L. A 34 amino acid lunch biotic polypeptide having a broad host spectrum produced by the Lactis strain. Nisin is widely used in foods as a preservative. First, nisin is synthesized by the ribosome as a precursor. After subsequent enzymatic modification, the modified molecule rearranges through the cytoplasmic membrane and is processed into its mature form.

Expression of the nucleic acid sequence of interest can be induced by the addition of nisin when the gene of interest is placed behind the inducible promoter PnisA of the nisin system.

The gene expression system controlled by nisin preferably allows for high protein yields, however, it is dependent on external inducers. When used for administration to a subject, the addition of nisin is expensive, as it requires the provision of pharmaceutical grade nisin, which raises additional regulatory issues.

Since nisin is a polypeptide, it is also prone to protease degradation. Therefore, if continuous expression of the nucleic acid sequence of interest is intended, nisin is preferably repetitively fed to the subject after administration of the recombinant bacterium expressing the nucleic acid sequence of interest under the control of the nisin promoter. It must be.

The requirement to add nisin for continuous expression of the nucleic acid sequence of interest further limits the applicable routes of administration of each recombinant bacterium.

For example, systemic administration of recombinant bacteria expressing the nucleic acid sequence of interest under the control of the nisin promoter may, in addition, to achieve concentrations of nisin within the subject that induce expression of the nucleic acid sequence of interest. Will require administration of a significant amount of nisin to the subject. Moreover, the toxicity profile of nisin and the need for availability of pharmaceutical grade nisin must be taken into account.

The heterologous polypeptide is at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof, without negatively impacting the viability of each recombinant bacterium used prior to administration to the subject. It is an object of the present invention to provide a bacterial expression system comprising, and independently allowing expression of at least one nucleic acid sequence encoding at least one heterologous factor or complex thereof, which is a heterologous polypeptide.

The bacterial expression system should provide inducible expression of each heterologous factor, at least by administration to the subject.

The bacterial expression system is such that the heterologous polypeptide to the subject is at least one eukaryotic polypeptide, preferably over a protracted period of time, without the need to supply additional inducing factors. It should also provide easy administration of at least one beneficial factor or complex thereof that comprises at least one fragment, or a combination thereof, and is independently a heterologous polypeptide.

Moreover, the bacterial expression system should provide a controlled amount of each beneficial factor, which is used in medicine.

An object of the present invention is
a) At least one nucleic acid sequence that is functionally coupled to a chloride-inducible promoter of a prokaryotic system and encodes at least one heterologous factor, the heterologous factor being independently a heterologous polypeptide or complex thereof. ,
b) At least one prokaryotic regulator gene that controls the activity of the chloride-inducible promoter,
To be resolved by providing a recombinant bacterium according to claim 1, comprising.
Heterologous polypeptides include at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof.

Preferably, the recombinant bacterium of the present invention is used for medical treatment.

Preferred embodiments of recombinant bacteria are disclosed in any one of Dependent Claims 3 to 13, 15 or 17 to 23.

Further, an object of the present invention is
a) At least one nucleic acid sequence that is functionally coupled to a chloride-inducible promoter of a prokaryotic system and encodes at least one heterologous factor, the heterologous factor being independently a heterologous polypeptide or complex thereof. ,
b) At least one prokaryotic regulator gene that controls the activity of the chloride-inducible promoter,
Resolved by providing a recombinant nucleic acid according to claim 2, comprising.
Heterologous polypeptides include eukaryotic polypeptides, at least one fragment thereof, or a combination thereof.

Preferably, the recombinant nucleic acid of the invention, preferably a plasmid, is used in the method for producing the recombinant bacterium of the invention.

Preferred embodiments of recombinant nucleic acids are disclosed in any one of Dependent Claims 4-11 or 14-16.

24. An object of the present invention further comprises a recombinant bacterium according to any one of claims 1, 3 to 13, 15, or 17 to 21, and at least one pharmaceutically acceptable excipient. It is solved by providing a pharmaceutical composition according to the above.

Preferably, the pharmaceutical composition of the present invention is used medically, preferably for the treatment of chronic inflammatory wounds or degenerative conditions, or for the treatment of tumors, preferably malignant tumors.

Preferred embodiments of pharmaceutical compositions are disclosed in any one of Dependent Claims 27 or 28.

Further, an object of the present invention is
a) Recombinant bacteria according to any one of claims 1, 3 to 13, 15 or 17 to 21, capable of expressing at least one heterologous factor under the control of a chloride-inducible promoter of a prokaryotic system.
b) At least one inducing factor, including chloride ion,
To be solved by a kit according to claim 25 for medical use, including.

Preferred embodiments of the kit are disclosed in any one of Dependent Claims 28-30.

Further, an object of the present invention is
a) Recombinant bacteria according to any one of claims 1, 3 to 13, 15 or 17 to 21, capable of expressing at least one heterologous factor under the control of a chloride-inducible promoter of a prokaryotic system.
Is settled by the medical device according to claim 26.

Preferred embodiments of the kit are disclosed in Dependent Claim 28.

It is an object of the present invention to further prepare a reprepared medium according to claim 31, which comprises chloride ions for repreparing a recombinant bacterium according to any one of claims 1, 3 to 13, 15, or 17 to 23. Resolved by use.

The present inventors have used recombinant bacteria containing the nucleic acid sequences mentioned above for medical treatment, preferably for the treatment of chronic inflammatory wounds or degenerative conditions, or for the treatment of tumors, preferably malignant tumors. It has been found that suitable prophylactic and / or therapeutic factors can be provided for subjects in need thereof without additional administration of exogenous inducing factors.

We further provide a sufficiently high concentration of chloride ion to the extracellular tissue environment or body fluid of interest to be functionally coupled to a chloride-inducible promoter of a prokaryotic system and at least one. It has been found to initiate and / or maintain expression of at least one nucleic acid sequence encoding a heterologous factor.

Suitable body fluids are, for example, extracellular fluids such as interstitial fluids, intravascular fluids such as blood, plasma, and serum, cerebrospinal fluid, peritoneal fluid, urine, tears, and lymph.

Moreover, the intracellular fluid of some mammalian phagocytic cells, such as neutrophils and monocytes, preferably has a sufficiently high intracellular chloride ion concentration and induces chloride in the prokaryotic system. Initiates and / or maintains expression of at least one nucleic acid sequence that is functionally coupled to the sex promoter and encodes at least one heterologous factor.

However, in the intracellular fluid of other mammalian cells, the intracellular chloride ion concentration is preferably insufficient for protein production, and therefore the recombinant bacterium of the present invention would enter the cytoplasm of these cells. Intracellular chloride concentration is characterized by safety features of expression of at least one nucleic acid sequence that is functionally coupled to a prokaryotic chloride-inducible promoter and encodes at least one heterologous factor. Will prevent initiation and / or maintenance.

These findings enable the provision of recombinant bacteria that can be designed as a single pharmaceutical entity, preferably based on bacteria genetically engineered to produce at least one heterologous factor, more preferably non-pathogenic lactic acid bacteria. To.

According to the invention, the term "heterologous factor" means a factor that is not naturally present in or is not expressed by the bacterium used, preferably a polypeptide, or a complex thereof.

This is generally used when referring to "heterogeneous factors (singular or plural)" or when referring to specific "heterologous factors (singular or plural)" such as FGF-2, IL-4, CSF-1. The term is intended to also include its functional analog (s).

According to the invention, the term "functional analog" of a factor is a drug that binds to the same receptor (s) to the same receptor (s) and preferably activates the same second messenger in the target cell. means.

Preferably, when each heterologous factor is a polypeptide or complex thereof, the "functional analog" of at least one heterologous factor is at least 50%, preferably at least 80%, even more preferably at least 90%. , More preferably at least 93%, even more preferably at least 95%, even more preferably at least 97% of the amino acid sequence sequence identity.

Preferably, when each heterologous factor is ribonucleic acid, the "functional analog" of at least one heterologous factor is at least 80%, more preferably at least 90%, even more preferably at least 93%, further. It preferably has at least 95%, more preferably at least 97%, sequence identity of the ribonucleic acid sequence.

"Functional analogs" can also be referred to as biosimilars.

Recombinant bacteria of the invention comprising at least one nucleic acid sequence referred to above by induction with chloride ions at least one by transcribing at least one nucleic acid sequence and preferably by translating. It can produce two heterologous factors.

In the presence of chloride ions, the recombinant bacterium of the invention can preferably deliver at least one heterologous factor according to claim 1, eg, to diseased tissue, thereby beneficial. Mediates and / or allows healing of the subject. Preferably, the recombinant bacterium of the invention releases at least one heterologous factor after administration to the subject.

In the presence of chloride ions, the recombinant bacterium of the invention preferably further provides a constant release of at least one heterologous factor, more preferably after administration to a subject in need thereof.

Thereby, a significantly improved, safer and more cost-effective treatment for subjects suffering from medical conditions, such as preferably chronic inflammatory wounds or degenerative conditions, or tumors, preferably malignant tumors. Options are available.

Preferably, the at least one heterologous factor exercises at least one bioactive function after release from the bacterium, supports healing of the subject, and / or prevents deterioration of the medical condition.

Furthermore, at least one heterologous factor may have prophylactic and / or therapeutic effects after release from the bacterium, which exercises, for example, paracrine and / or endocrine activity that impacts local or systemic metabolism. And / or by controlling the activity of cells in the body, and / or by impacting the viability, growth, and differentiation of various cells of the body, and / or against injury and / or infection. By impacting the immune regulation or induction of the acute inflammatory response.

The recombinant bacterium of the present invention is:
a) At least one nucleic acid sequence that is functionally coupled to a chloride-inducible promoter of a prokaryotic system and encodes at least one heterologous factor, the heterologous factor being independently a heterologous polypeptide or complex thereof. ,
b) At least one prokaryotic regulator gene that controls the activity of the chloride-inducible promoter,
Including
Heterologous polypeptides include at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof.

The term "protozoan promoter" is known to those of skill in the art and preferably initiates transcription of a particular gene by providing a binding site for RNA polymerase and / or at least one transcription factor that recruits RNA polymerase. Refers to the nucleic acid sequence that controls.

The prokaryotic promoter is preferably located near the transcription start side of the gene, more preferably on the same strand of the gene to be transcribed, and upstream, which is in the 5'region of the same strand. The direction.

The term "functionally coupled to" means that transcription of at least one nucleic acid sequence encoding at least one heterologous factor is preferably initiated and controlled by the respective prokaryotic promoter. ..

Preferably, the prokaryotic promoter used according to the invention to initiate and control transcription of at least one nucleic acid sequence encoding at least one heterologous factor is a prokaryotic promoter that is inducible by chloride ions. be.

More preferably, the activity of the prokaryotic chloride-inducible promoter depends on the concentration of chloride ions in the environment of the recombinant bacterium, such as the reprepared medium or body fluid after application to the subject.

The term "activity" of a prokaryotic chloride-inducible promoter preferably refers to the amount of at least one heterologous factor expressed from at least one nucleic acid sequence functionally coupled to the promoter.

Preferably, the term "activity" of a prokaryotic chloride-inducible promoter refers to the amount of protein (s) obtained from transcription of at least one nucleic acid sequence encoding at least one heterologous factor.

Preferably, at least one heterologous factor is produced from the transcribed mRNA, especially by translation of the mRNA.

The activity of the promoter used according to the invention, preferably to initiate and control transcription of at least one nucleic acid sequence encoding at least one heterologous factor, is further controlled by at least one prokaryotic regulator gene. ..

The term "regulator gene" is known to those of skill in the art and refers to a nucleic acid sequence, preferably a gene, involved in controlling the expression of one or more other genes. The control sequence, preferably encoding the control gene, is preferably located 5'with respect to the transcription start site of the gene to be controlled. The control sequence can also be located 3'with respect to the transcription initiation site, or also at a distant site on the chromosome.

The regulator gene may be preferably located on the operon, adjacent to or far from it, and even more preferably within the same bacterial cell.

In a preferred embodiment, the regulator gene encodes a regulator protein such as a repressor protein or an activator protein, more preferably the expression of the regulator protein is a constitutive promoter or a controlled preferably inducible promoter. It is initiated and controlled by an individual prokaryotic system promoter selected from at least one of the above.

More preferably, the expression of the regulator protein is controlled by a constitutive prokaryotic promoter, whereby the recombinant bacterium of the invention has a sufficient amount of regulator protein to control the activity of the chloride-inducible promoter. It can be provided intracellularly.

The repressor protein preferably binds to the operator or promoter and prevents RNA polymerase from transcribing RNA. At least one inducing factor can preferably cause the repressor protein to change shape or otherwise become unable to bind to DNA, allowing RNA polymerase to initiate and / or continue transcription.

An activator protein such as GadR preferably binds to a site on the DNA molecule, preferably near a promoter to be controlled by the regulator protein, allowing transcription and / or enhancing the rate of transcription. ..

According to the present invention, at least one nucleic acid sequence that is functionally coupled to a prokaryotic chloride-inducible promoter and encodes at least one heterologous factor and at least one that controls the activity of the chloride-inducible promoter. The two prokaryotic regulator genes are respectively located in the same bacterial cell of the recombinant bacterium of the present invention, preferably in each bacterial cell.

Preferably, at least one nucleic acid sequence that is functionally coupled to a prokaryotic chloride-inducible promoter and encodes at least one heterologous factor and at least one prokaryotic that controls the activity of the chloride-inducible promoter. Each bioregulatory factor gene is independently located on a chromosome and / or at least one plasmid in the same bacterial cell of the recombinant bacterium.

For example, at least one nucleic acid sequence that is functionally coupled to a prokaryotic chloride-inducible promoter and encodes at least one heterologous factor is a chromosome and in at least one bacterial cell of the recombinant bacterium of the invention. / Or located on at least one of the plasmids. Independently, at least one prokaryotic regulator gene is preferably located on at least one of the chromosomes and / or plasmids of the same bacterial cell of the recombinant bacterium.

Preferably, at least one nucleic acid sequence that is functionally coupled to a prokaryotic chloride-inducible promoter and encodes at least one heterologous factor and at least one prokaryotic that controls the activity of the chloride-inducible promoter. Biological regulator genes are located on the same recombinant nucleic acid molecule, which is at least one of the chromosomes and / or plasmids, respectively.

More preferably, at least one nucleic acid sequence that is functionally coupled to a prokaryotic chloride-inducible promoter and encodes at least one heterologous factor and at least one that controls the activity of the chloride-inducible promoter. The prokaryotic regulator genes, at least both, are located on the recombinant nucleic acids of the invention, more preferably on recombinant plasmids.

The recombinant nucleic acid of the present invention, preferably the recombinant plasmid, is
a) At least one nucleic acid sequence that is functionally coupled to a chloride-inducible promoter of a prokaryotic system and encodes at least one heterologous factor, the heterologous factor being independently a heterologous polypeptide or complex thereof. ,
b) At least one prokaryotic regulator gene that controls the activity of the chloride-inducible promoter,
Including
Heterologous polypeptides include eukaryotic polypeptides, at least one fragment thereof, or a combination thereof.

The term "plasmid" is known to those of skill in the art and refers to a preferably circular, more preferably double-stranded DNA molecule in a bacterial cell that can be physically separated from chromosomal DNA and replicate independently.

That is, the recombinant nucleic acid of the present invention is preferably a circular, more preferably double-stranded DNA molecule.
a) At least one nucleic acid sequence that is functionally coupled to a chloride-inducible promoter of a prokaryotic system and encodes at least one heterologous factor, the heterologous factor being independently a heterologous polypeptide or complex thereof. ,
b) At least one prokaryotic regulator gene that controls the activity of the chloride-inducible promoter,
Including
Heterologous polypeptides include eukaryotic polypeptides, at least one fragment thereof, or a combination thereof.

The recombinant bacterium of the invention comprises at least one copy of the recombinant nucleic acid of the invention, preferably a recombinant plasmid.

At least one nucleic acid sequence that is functionally coupled to a prokaryotic chloride-inducible promoter encodes at least one heterologous factor.

Preferably, each at least one nucleic acid sequence encodes one or more factors (s) that are not naturally present in the bacterium used or are not expressed by the bacterium.

More preferably, at least one nucleic acid sequence encodes 1, 2, 3, 4, or more factors, each of which is not naturally present in the bacterium used or is not expressed by the bacterium and is independent. And a heterologous polypeptide or a complex thereof, preferably a heterologous polypeptide or a complex thereof.

The term "complex" refers to a protein complex of two or more polypeptide chains, which may be referred to as a "subunit", preferably at least one non-covalent protein-protein interaction. Associated or linked by, for example, hydrogen bonds, ionic interactions, van der Waals forces, and / or hydrophobic bonds, and / or by at least one covalent protein-protein bond that is not a peptide bond, such as a disulfide bond. ..

The subunits of a multimeric protein complex can be identical, such as a homomultimeric protein complex, or different, such as a heteromultimeric protein complex.

Preferably, the complex of the heterologous polypeptide is formed before and / or after the release of at least one heterologous polypeptide from the recombinant bacterium of the invention.

For example, the recombinant bacterium of the invention expresses one heterologous polypeptide, which forms a homomultimeric protein complex with two or more identical subunits after release from the bacterium.

Alternatively, the recombinant bacteria of the invention express two or more heterologous polypeptides, which are different from each other and are heteromultimeric protein complexes having two or more different subunits after release from the bacterium. To form.

Preferably, the at least one heterologous factor has a therapeutic and / or preventive effect in the subject, preferably after application of the recombinant bacterium of the invention to the subject.

In a more preferred embodiment of the invention, each of the heterologous factors comprises or comprises at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof, or a heterologous polypeptide thereof. It is a complex.

More preferably, the term "fragment of a eukaryotic polypeptide" refers to a bioactive fragment of a eukaryotic polypeptide.

Preferably, the recombinant bacterium of the invention expresses at least one heterologous polypeptide or complex thereof after application to the subject. More preferably, the recombinant bacterium of the invention releases, preferably secretes, at least one heterologous polypeptide or complex thereof into the surrounding environment, eg, the site of application and / or body fluid of interest.

The at least one heterologous polypeptide or complex thereof can itself have a biological effect on at least one cell of interest, which, for example, stimulates or inhibits cell growth, proliferation, and / or cell differentiation. By inducing or suppressing apoptosis, by activating or inhibiting the immune system, by controlling metabolism, by controlling cell migration, and / or by producing and / or the subject's endogenous factors. By controlling the release, thereby treating and / / in the subject, preferably after application of the recombinant bacterium of the invention to the subject, preferably after release from the recombinant bacterium of the invention, more preferably after secretion. Or mediate the preventive effect.

The therapeutic and / or preventive effect in a subject is at least one heterologous polypeptide or at least one heterologous polypeptide against at least one endogenous or exogenous target molecule present in the subject, such as a bacterial antigen, a viral antigen, a tumor antigen, and / or an endogenous polypeptide. It can also be mediated by the binding of the complex.

For example, at least one heterologous polypeptide or complex thereof is an antibody and / or at least one bioactive fragment thereof, which is released from the bacterium after application of the recombinant bacterium of the invention to a subject and at least afterwards. It binds to one target molecule, preferably a foreign antigen, such as a part of a bacterial cell or virus, or an endogenous antigen or polypeptide, but otherwise they have harmful effects such as an overactive immune response. Will mediate.

By binding to at least one target molecule present in the subject, the at least one heterologous polypeptide or complex thereof preferably reduces the amount of the molecule in the subject and / or the biological activity of the molecule in the subject. And / or prevent, for example, by reducing or inhibiting the enzymatic activity of the molecule and / or by reducing or inhibiting the binding of the molecule to an endogenous receptor.

Preferably, the at least one heterologous factor is a heterologous polypeptide, preferably at least one fragment thereof having at least 5, preferably at least 7 amino acids linked by peptide bonds, and / or a complex thereof.

Examples of suitable polypeptides include eukaryotic species capable of acting locally and / or systemically, preferably mammalian species, more preferably polypeptides from humans, precursors thereof, fragments thereof, and Including those combinations.

More preferably, the at least one heterologous polypeptide is a growth factor, cytokine, chemokine, enzyme, polypeptide hormone, neuropeptide, antibody, cell surface receptor from a eukaryotic species, preferably a mammalian species, even more preferably a human. Intrabodies, cofactors, transcription factors, adhesive molecules, tumor antigens, precursors thereof, preferably bioactive fragments thereof, which may also be referred to as bodies, soluble receptors, receptor ligands, intracellular antibodies, and theirs. Selected from a group of combinations.

Suitable tumor antigens are known to those of skill in the art, preferably Cheever, M. et al. A. et al. (2009). It is disclosed in CCR-09-0737).

Of course, functional analogs of the polypeptides mentioned above or mentioned below or biosimilars thereof may also be used within the scope of the claimed invention.

Growth factors are preferably polypeptides that can stimulate cell growth, proliferation, healing, and / or cell differentiation.

Preferably, the growth factors are fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth. It is selected from the group consisting of factor beta (TGF-beta), nerve growth factor (NGF), activin, their functional analogs, their biosimilars, and mixtures thereof.

Fibroblast growth factors are a family of growth factors involved in angiogenesis, wound healing, and various endocrine signaling pathways. In humans, 22 members of the FGF family, FGF-14 to FGF-14 and FGF-16 to FGF-23, have been identified and can be used in the present invention. FGF-1 to FGF-10 bind to the fibroblast growth factor receptor (FGFR).

In a preferred embodiment, the fibroblast growth factor is FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF- 10, and mixtures thereof, more preferably FGF-1, FGF-2, FGF-7, FGF-10, and mixtures thereof, more preferably FGF-2, FGF-7, their functional analogs, among them. It is selected from the group consisting of biosimilars and mixtures thereof, more preferably FGF-2.

For example, FGF-1 and FGF-2 can stimulate angiogenesis and are mitogenic for some cell types present at the site of inflammatory skin disorders, including fibroblasts and keratinocytes. Moreover, FGF-7 can stimulate wound re-epithelialization in a paracrine manner.

The nucleic acid sequence of human fibroblast growth factor 2 (hFGF-2) mRNA is available at NCBI Accession No. NM_002006.4. Each amino acid sequence of the AUG isomer is available in NCBI accession number NP_0019977.5 and UniProt accession number P09038 version 182.

The precursor comprises a propeptide spanning amino acids 1 to 142 of the precursor and a mature human fibroblast growth factor 2 peptide spanning amino acids 143 to 288 of the precursor.

In a preferred embodiment, fibroblast growth factor 2 comprises one or at least one of the amino acid sequences of SEQ ID NOs: 5-7. The amino acid sequences of SEQ ID NOs: 5 to 7 are shown in FIGS. 5a to 5c, respectively.

Insulin-like growth factor (IGF) is a protein with high sequence similarity to insulin. Insulin-like growth factor contains two proteins, IGF-1 and IGF-2, which can be used in the present invention.

The epidermal growth factor (EGF) family is a protein with highly similar structural and functional characteristics, including epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), and transforming growth factor. -Α (TGF-α), Amphiregulin (AR), Epiregulin (EPR), Epigen (EPGN), Betacellulin (BTC), Newregrin-1 (NRG1), Newregrin-2 (NRG2), Newregrin- 3 (NRG3), and neuregulin-4 (NRG4), preferably epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), Epiregulin (EPR), Epigen (EPGN), and Betacellulin (BTC), more preferably epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), and transforming growth factor-α. Includes (TGF-α), which can be used in the present invention.

Transforming growth factor-α (TGF-α), preferably human transforming growth factor-α (hTGF-α), can be produced by macrophages, brain cells, and keratinocytes. hTGF-α induces epithelial development. hTGF-α and hEGF bind to the epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) of the same receptor. When TGF-α binds to EGFR, it can initiate multiple cell proliferation events involving wound healing.

Human transforming growth factor-α exists as at least five isoforms produced by alternative splicing.

The amino acid sequence of the human transforming growth factor alpha isoform 1 precursor is available at NCBI Accession No. NP_003227.1. Each nucleic acid sequence of the mRNA is available at NCBI Accession No. NM_003236.2.

The precursors of human transforming growth factor alpha isoform 1 are signal peptides spanning amino acids 1 to 23 of the precursor, protransforming growth factor alpha isoform 1 spanning amino acids 24 to 160 of the precursor, and amino acids of the precursor. Includes mature transforming growth factor alpha peptides spanning 40-89.

The amino acid sequence of the human transforming growth factor alpha isoform 2 precursor is available at NCBI Accession No. NP_001093161.1. Each nucleic acid sequence of the mRNA is available at NCBI Accession No. NM_0010999691.1.

The amino acid sequence of the human transforming growth factor alpha isoform 3 precursor is available at NCBI Accession No. NP_001295087.1. Each nucleic acid sequence of the mRNA is available at NCBI Accession No. NM_001308158.1.

The amino acid sequence of the human transforming growth factor alpha isoform 4 precursor is available at NCBI Accession No. NP_001295088.1. Each nucleic acid sequence of the mRNA is available at NCBI Accession No. NM_001308159.1.

The amino acid sequence of the human transforming growth factor alpha isoform 5 precursor is available at NCBI Accession No. AAF05090.1. Each nucleic acid sequence of the mRNA is available at NCBI Accession No. AF149097.1.

Amphiregulin (AREG), preferably human ampphiregulin (hAREG), is another ligand for the EGF receptor. Human ampphiregulin is an autocrine growth factor and mitogen for a wide range of target cells, including astrocytes, Schwann cells, and fibroblasts. Human amphiregulin promotes the growth of epithelial cells.

The amino acid sequence of the human amphiregulin precursor is available at NCBI Accession No. NP_001648.1. Each nucleic acid sequence of the mRNA is available at NCBI Accession No. NM_001657.3.

Epiregulin (EPR), preferably human epiregulin (hEPR), is a ligand for the EGF receptor, which can stimulate cell proliferation and / or angiogenesis.

The amino acid sequence of the human epiregulin precursor is available at NCBI Accession No. NP_001423.1. Each nucleic acid sequence of the mRNA is available at NCBI Accession No. NM_001432.1.

Epigen (EPGN), preferably human epigen (hEPGN), promotes the growth of epithelial cells. Human epigens exist as at least seven isoforms produced by alternative splicing.

The amino acid sequence of the human epigen isoform 1 precursor is available at NCBI Accession No. NP_001257918.1. Each nucleic acid sequence of the mRNA is available at NCBI Accession No. NM_0012709989.1.

The amino acid sequences of the human epigen isoforms 1-7 precursors are also available in UniProt accession number Q6UW88 version 101.

Betacellulin (BTC), preferably human betacellulin (hBTC), also binds to epidermal growth factor receptors and is by a wide range of adult tissues and in many cultured cells, including smooth muscle cells and epithelial cells. It is a growth factor that is synthesized. The amino acid sequence of the human probetacellulin precursor is available at NCBI Accession No. NP_001720.1. Each nucleic acid sequence of the mRNA is available at NCBI Accession No. NM_001729.1.

Insulin-like growth factor 1 (IGF-1) is also called somatomedin C. The nucleic acid sequence of human IGF-1 mRNA is available at NCBI Accession No. NM_000618.2. Each amino acid sequence is available in NCBI accession number NP_0000609.1 and UniProt accession number P05019 version 178.

The nucleic acid sequence of human insulin-like growth factor 2 (hIGF-2) mRNA is available at NCBI Accession No. NM_000612.4. The respective amino acid sequences of the human insulin-like growth factor 2 precursor are available in NCBI accession number NP_000603.1 and UniProt accession number P01344 version 192.

The family of vascular endothelial growth factor (VEGF) is a group of growth factors, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PGF), which are used in the present invention. Can be.

In a preferred embodiment, the vascular endothelial growth factor is vascular endothelial growth factor A (VEGF-A). VEGF-A can induce angiogenesis, angiogenesis, and endothelial cell growth.

The nucleic acid sequence of the human vascular endothelial growth factor A (hVEGF-A) mRNA is available at NCBI Accession No. NM_001025366.1. The respective amino acid sequences of human vascular endothelial growth factor A are available in NCBI Accession No. NP_001020537.2. And UniProt Accession No. P15692 Version 197.

Platelet-derived growth factor (PDGF) regulates cell growth and division. Human platelet-derived growth factor (hPDGF) has four subunits, PDGF-A, PDGF-B, PDGF-C, and PDGF-D, which are either homo or heterodimers of each subunit. However, these can be used in the present invention.

Preferably, the platelet-derived growth factor is PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, PDGF-DD, or a mixture thereof.

More preferably, platelet-derived growth factor is a dimeric protein composed of two PDGF-A subunits, a dimeric protein composed of two PDGF-B subunits, PDGF-A subunit and PDGF-. A dimeric protein composed of B subunits, or a mixture thereof.

The nucleic acid sequence of the mRNA of the human platelet-derived growth factor subunit A (hPDGF-A) is available at NCBI Accession No. NM_002607.4. Each amino acid sequence is available in NCBI accession number NP_002598.4 and UniProt accession number P04085 version 159.

The nucleic acid sequence of the mRNA of human platelet-derived growth factor subunit B (hPDGF) is available at NCBI Accession No. NM_002608.1. The respective amino acid sequences of the human platelet-derived growth factor subunit precursor are available in NCBI Accession No. NP_002599.1 and UniProt Accession No. P01127 Version 181.

Hepatocyte growth factor (HGF) is a growth factor that is secreted by mesenchymal cells and acts primarily on epithelial and endothelial cells but also on hematopoietic progenitor cells and can be used in the present invention.

The nucleic acid sequence of human hepatocyte growth factor (hHGF) mRNA is available at NCBI Accession No. NM_0000601.3. The respective amino acid sequences of human hepatocyte growth factor precursors are available in NCBI Accession No. NP_000592.3 and UniProt Accession No. P14210 version 186.

Transforming growth factor β (TGF-β), preferably human transforming growth factor β (hTGF-β), is a cytokine secreted by many cell types, including macrophages.

TGF-β exists as at least three isoforms TGF-β1, TFG-β2, and TGF-β3, which can be used in the present invention.

Human transforming growth factor β1 is a secretory protein that is cleaved into a latently associated peptide (LAP) and a mature TGF-β1 peptide. The mature peptide can form either a TGF-β1 homodimer or a heterodimer with other TGF-β family members.

Nucleic acid sequences of human transforming growth factor β1 precursor mRNA can be obtained by NCBI Accession No. NM_000660.4. Each amino acid sequence is available at NCBI Accession No. NP_000651.3. Or UniProt Accession No. P01137 Version 199.

Transforming growth factor β2 (TGF-β2), preferably human transforming growth factor β2 (hTGF-β2), is a multifunctional cytokine that controls proliferation, differentiation, adhesion, and migration of many cell types.

Alternatively, splicing transcript variants of the human transforming growth factor β2 gene encoding two different isoforms have been identified.

The nucleic acid sequence of the mRNA of the human transforming growth factor beta 2 isoform 1 precursor is available at NCBI Accession No. NM_0011355999.3. Each amino acid sequence is available at NCBI Accession No. NP_001122971.1.

The nucleic acid sequence of the human transforming growth factor β2 isoform 2 precursor mRNA is available at NCBI Accession No. NM_003238.3. Each amino acid sequence is available at NCBI Accession No. NP_003229.1. The amino acid sequence of transforming growth factor β2 is also available in UniProt accession number P61812 version 128.

Transforming growth factor β3 (TGF-β3), preferably human transforming growth factor β3 (hTGF-β3), is a secretory cytokine involved in embryogenesis and cell differentiation.

The nucleic acid sequence of the human transforming growth factor β3 precursor protein mRNA is available at NCBI Accession No. NM_003239.3. The corresponding amino acid sequences are available in NCBI accession number NP_003230.1 and UniProt accession number P10600 version 170.

Activin is a disulfide-linked dimeric protein originally purified from gonad fluid as a protein that stimulates pituitary follicle-stimulating hormone (FSH) release. Activin protein has a wide range of biological activities including roles in mesoderm induction, nervous system cell differentiation, bone remodeling, hematopoiesis, and reproductive physiology.

Activin is a homodimer or heterodimer of various beta subunit isoforms, and activin is a unique alpha subunit and four beta subunits, beta A, beta B, beta C, and beta. It is a heterodimer with one of E.

Cytokines are preferably polypeptides that are involved as immunomodulators in autocrine signaling, paracrine signaling, and endocrine signaling.

Preferably, the cytokine is selected from the group consisting of interferon, interleukin, lymphokine, tumor necrosis factor, colony stimulating factor, their functional analogs, their biosimilars, and mixtures thereof.

Preferably, interferon, more preferably human interferon, is interferon alpha (IFN-α), interferon beta (IFN-β), interferon epsilon (IFN-ε), interferon kappa (IFN-κ), interferon gamma (IFN-γ). ), Interferon omega (IFN-ω), interferon lambda (IFN-λ), their functional analogs, their biosimilars, and mixtures thereof.

Interferon alpha, preferably human interferon alpha, is preferably interferon alpha-1 (IFN-α1), interferon alpha-2 (IFN-α2), interferon alpha-4 (IFN-α4), interferon alpha-5 (IFN-α4). α5), Interferon Alpha-6 (IFN-α6), Interferon Alpha-7 (IFN-α7), Interferon Alpha-8 (IFN-α8), Interferon Alpha-10 (IFN-α10), Interferon Alpha-13 (IFN- α13), interferon alpha-14 (IFN-α14), interferon alpha-16 (IFN-α16), interferonalpha-17 (IFN-α17), interferonalpha-21 (IFN-α21), their functional analogs, they Biosimilars and mixtures thereof, preferably interferon alpha-2 (IFN-α2), functional analogs thereof, biosimilars thereof, and mixtures thereof.

The nucleic acid sequence of the human interferon alpha 1 (IFN-α1) mRNA is available at NCBI Accession No. NM_024013.2. The nucleic acid sequence of the human interferon alpha 13 (IFN-α13) mRNA is available at NCBI Accession No. NM_006900.3.

Human interferons alpha-1 and alpha-13 have the same protein sequence. The respective amino acid sequences of the human interferon alpha-1 / 13 precursor are available in NCBI accession numbers NP_076918.1 and NP_008831.3. And UniProt accession number P01562 version 180.

The precursors of human interferon alpha-1 / 13 include a signal peptide spanning amino acids 1 to 23 of the precursor and mature interferon alpha-2 spanning amino acids 24 to 189 of the precursor.

The nucleic acid sequence of human interferon alpha 2 (IFN-α2) mRNA is available at NCBI Accession No. NM_000605.3. The respective amino acid sequences of the human interferon alpha-2 precursor are available in NCBI accession number NP_000596.2 and UniProt accession number P01563 version 176.

Precursors for human interferon alpha-2 include signal peptides that span amino acids 1 to 23 of the precursor and mature interferon alpha-2 that spans amino acids 24 to 188 of the precursor.

More preferably, interferon alpha-2 comprises one or at least one of the amino acid sequences of SEQ ID NOs: 28-30, which are also illustrated in FIGS. 14a-14c, respectively.

The nucleic acid sequence of the human interferon alpha 4 (IFN-α4) mRNA is available at NCBI Accession No. NM_021068.2. The respective amino acid sequences of the human interferon alpha-4 precursor are available in NCBI accession number NP_0666546.1 and UniProt accession number P05014 version 168.

Precursors for human interferon alpha-4 include signal peptides that span amino acids 1 to 23 of the precursor and mature interferon alpha-4 that spans amino acids 24 to 189 of the precursor.

The nucleic acid sequence of the human interferon alpha 5 (IFN-α5) mRNA is available at NCBI Accession No. NM_002169.2. The respective amino acid sequences of the human interferon alpha-5 precursor are available in NCBI accession number NP_002160.1 and UniProt accession number P01569 version 158.

Precursors for human interferon alpha-5 include signal peptides that span amino acids 1 to 21 of the precursor and mature interferon alpha-5 that spans amino acids 22 to 189 of the precursor.

The nucleic acid sequence of human interferon alpha 6 (IFN-α6) mRNA is available at NCBI Accession No. NM_021002.2. The respective amino acid sequences of the human interferon alpha-6 precursor are available in NCBI accession number NP_066282.1 and UniProt accession number P05013 version 158.

Precursors for human interferon alpha-6 include signal peptides spanning amino acids 1-20 of the precursor and mature interferon alpha-6 spanning amino acids 21-189 of the precursor.

The nucleic acid sequence of the human interferon alpha 7 (IFN-α7) mRNA is available at NCBI Accession No. NM_021057.2. The respective amino acid sequences of the human interferon alpha-7 precursor are available in NCBI accession number NP_066401.2. and UniProt accession number P01567 version 160.

Precursors for human interferon alpha-7 include signal peptides that span amino acids 1 to 23 of the precursor and mature interferon alpha-7 that spans amino acids 24 to 189 of the precursor.

The nucleic acid sequence of human interferon alpha 8 (IFN-α8) mRNA is available at NCBI Accession No. NM_002170.3. The respective amino acid sequences of the human interferon alpha-8 precursor are available in NCBI accession number NP_002161.2. And UniProt accession number P32881 version 157.

Precursors for human interferon alpha-8 include signal peptides that span amino acids 1 to 23 of the precursor and mature interferon alpha-8 that spans amino acids 24 to 189 of the precursor.

The nucleic acid sequence of human interferon alpha 10 (IFN-α10) mRNA is available at NCBI Accession No. NM_002171.2. The respective amino acid sequences of the human interferon alpha-10 precursor are available in NCBI accession number NP_002162.1 and UniProt accession number P01566 version 160.

Precursors for human interferon alpha-10 include signal peptides that span amino acids 1 to 23 of the precursor and mature interferon alpha-10 that spans amino acids 24 to 189 of the precursor.

The nucleic acid sequence of the human interferon alpha 14 (IFN-α14) mRNA is available at NCBI Accession No. NM_002172.2. The respective amino acid sequences of the human interferon alpha-14 precursor are available in NCBI accession number NP_002163.2. And UniProt accession number P01570 version 172.

Precursors for human interferon alpha-14 include signal peptides that span amino acids 1 to 23 of the precursor and mature interferon alpha-14 that spans amino acids 24 to 189 of the precursor.

The nucleic acid sequence of the human interferon alpha 16 (IFN-α16) mRNA is available at NCBI Accession No. NM_002173.3. The respective amino acid sequences of the human interferon alpha-16 precursor are available in NCBI accession number NP_002164.1 and UniProt accession number P05015 version 161.

Precursors for human interferon alpha-16 include signal peptides that span amino acids 1 to 23 of the precursor and mature interferon alpha-16 that spans amino acids 24 to 189 of the precursor.

The nucleic acid sequence of the human interferon alpha 17 (IFN-α17) mRNA is available at NCBI Accession No. NM_021268.2. The respective amino acid sequences of the human interferon alpha-17 precursor are available in NCBI accession number NP_067091.1 and UniProt accession number P01571 version 162.

Precursors for human interferon alpha-17 include signal peptides that span amino acids 1 to 23 of the precursor and mature interferon alpha-17 that spans amino acids 24 to 189 of the precursor.

The nucleic acid sequence of the human interferon alpha 21 (IFN-α21) mRNA is available at NCBI Accession No. NM_002175.2. The respective amino acid sequences of the human interferon alpha-21 precursor are available in NCBI accession number NP_002166.2 and UniProt accession number P01568 version 171.

Precursors for human interferon alpha-21 include signal peptides that span amino acids 1 to 23 of the precursor and mature interferon alpha-21 that spans amino acids 24 to 189 of the precursor.

The nucleic acid sequence of human interferon beta 1 (IFN-β1) mRNA is available at NCBI Accession No. NM_002176.3. The respective amino acid sequences of the human interferon beta precursors are available in NCBI Accession No. NP_002167.1 and UniProt Accession No. P015774 Version 196.

Precursors for human interferon beta include signal peptides spanning amino acids 1 to 21 of the precursor and mature interferon beta spanning amino acids 22 to 187 of the precursor.

The nucleic acid sequence of human interferon gamma (IFN-γ) mRNA is available at NCBI Accession No. NM_000619.2. The respective amino acid sequences of the human interferon gamma precursor are available in NCBI accession number NP_0006100.2 and UniProt accession number P01579 version 205.

Precursors of human interferon gamma include signal peptides spanning amino acids 1 to 23 of the precursor, mature interferon gamma spanning amino acids 24 to 161 of the precursor, and propeptides spanning amino acids 162 to 166 of the precursor.

The nucleic acid sequence of the human interferon kappa (IFN-κ) mRNA is available at NCBI Accession No. NM_020124.2. The respective amino acid sequences of the human interferon kappa precursor are available in NCBI accession number NP_064509.2 and UniProt accession number Q9P0W0 version 120.

The precursors of human interferon kappa include a signal peptide spanning amino acids 1-27 of the precursor and a mature interferon gamma spanning amino acids 28-207 of the precursor.

The nucleic acid sequence of the human interferon epsilon (IFN-ε) mRNA is available at NCBI Accession No. NM_176891.4. The respective amino acid sequences of the human interferon epsilon precursor are available in NCBI accession number NP_795372.1 and UniProt accession number Q86WN2 version 124.

Precursors of human interferon epsilon include signal peptides spanning amino acids 1 to 21 of the precursor and mature interferon epsilon spanning amino acids 22 to 208 of the precursor.

The nucleic acid sequence of the human interferon omega 1 (IFN-ω1) mRNA is available at NCBI Accession No. NM_002177.2. The respective amino acid sequences of the human interferon omega precursor are available in NCBI accession number NP_002168.1 and UniProt accession number P05000 version 165.

Precursors of human interferon omega include signal peptides spanning amino acids 1 to 21 of the precursor and mature interferon omega spanning amino acids 22 to 195 of the precursor.

The human interferon lambda (IFN-λ) is preferably interferon lambda-1 (IFN-λ1), interferon lambda-2 (IFN-λ2), interferon lambda-3 (IFN-λ3), interferon lambda-4 (IFN- It is selected from the group consisting of λ4), their functional analogs, their biosimilars, and their mixtures.

The nucleic acid sequence of the human interferon lambda 1 (IFN-λ1) mRNA is available at NCBI Accession No. NM_172140.1. The respective amino acid sequences of the human interferon lambda-1 precursor are available in NCBI accession number NP_742152.1. And UniProt accession number Q8IU54 version 125.

Precursors for human interferon lambda-1 include signal peptides spanning amino acids 1-19 of the precursor and mature interferon lambda-1 spanning amino acids 20-200 of the precursor.

The nucleic acid sequence of the human interferon lambda 2 (IFN-λ2) mRNA is available at NCBI Accession No. NM_17238.1. The respective amino acid sequences of the human interferon lambda-2 precursor are available in NCBI accession number NP_7421500.1 and UniProt accession number Q8IZJ0 version 105.

The precursors of the human interferon lambda-2 precursor include a signal peptide spanning amino acids 1 to 25 of the precursor and a mature interferon lambda-2 spanning amino acids 26 to 200 of the precursor.

The nucleic acid sequence of human interferon lambda 3 (IFN-λ3) mRNA comprises two transcript variants. The mRNA sequence for human interferon lambda 3 transcript variant 1 is available at NCBI Accession No. NM_001346937.1. The mRNA sequence for human interferon lambda 3 transcript variant 2 is available at NCBI Accession No. NM_17239.3.

The respective amino acid sequences of the human interferon lambda-3 isoform 1 precursor are available at NCBI Accession No. NP_001333866.1. The respective amino acid sequences of the human interferon lambda-3 isoform 2 precursor are available in NCBI accession number NP_742151.2 and UniProt accession number Q8IZI9 version 113.

The precursor of human interferon lambda-3 isoform 2 includes a signal peptide spanning amino acids 1 to 21 of the precursor and mature interferon lambda-3 spanning amino acids 22 to 196 of the precursor.

The nucleic acid sequence of the human interferon lambda 4 (IFN-λ4) mRNA is available at NCBI Accession No. NM_0012762254.2. The respective amino acid sequences of the human interferon lambda-4 precursor are available in NCBI accession number NP_001263183.2 and UniProt accession number K9M1U5 version 24.

Precursors for human interferon lambda-4 include signal peptides spanning amino acids 1 to 21 of the precursor and mature interferon lambda-4 spanning amino acids 22 to 179 of the precursor.

The interleukin, more preferably the human interleukin, is preferably interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL). -4), Interleukin-5 (IL-5), Interleukin-6 (IL-6), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Interleukin-9 (IL) -9), Interleukin-10 (IL-10), Interleukin-11 (IL-11), Interleukin-12 (IL-12), Interleukin-13 (IL-13), Interleukin-14 (IL) -14), Interleukin-15 (IL-15), Interleukin-16 (IL-16), Interleukin-17 (IL-17), Interleukin-18 (IL-18), Interleukin-19 (IL) -19), Interleukin-20 (IL-20), Interleukin-21 (IL-21), Interleukin-22 (IL-22), Interleukin-23 (IL-23), Interleukin-24 (IL) -24), Interleukin-25 (IL-25), Interleukin-26 (IL-26), Interleukin-27 (IL-27), Interleukin-28 (IL-28), Interleukin-29 (IL) -29), interleukin-30 (IL-30), interleukin-31 (IL-31), interleukin-32 (IL-32), interleukin-33 (IL-33), interleukin 34 (IL- 34), Interleukin-36alpha (IL-36α), Interleukin-36beta (IL-36β), Interleukin-36gamma (IL-36γ), Interleukin-37 (IL-37), Cardiotrophin Like cytokine factor 1 (CLCF1), hairy neuronutrient factor (CNTF), leukemia inhibitor (LIF), oncostatin-M (OSM), interleukin receptor antagonist protein, interleukin-binding protein, their functional analogs , Their biosimilars, and mixtures thereof, preferably interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-18 (IL-18), their functional analogs, They are selected from the group consisting of biosimilars and mixtures thereof.

Preferably, interleukin-1 (IL-1), more preferably human interleukin-1, is interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), functional analogs thereof. , Their biosimilars, and mixtures thereof are selected.

The nucleic acid sequence of the human interleukin-1alpha (IL-1α) precursor mRNA is available at NCBI Accession No. NM_0005755.4. Each amino acid sequence of the precursor is available at NCBI Accession No. NP_000566.3.

The amino acid sequence of the human interleukin-1 alpha precursor is also available in UniProt accession number P01583 version 1908. The amino acid sequence comprises a propeptide spanning amino acids 1-112 of the interleukin-1alpha precursor and a mature interleukin-1alpha spanning amino acids 113-271 of the precursor.

The nucleic acid sequence of the human interleukin-2 precursor mRNA is available at NCBI Accession No. NM_000586.3. Each amino acid sequence is available at NCBI Accession No. NP_000577.2.

The amino acid sequence of the human interleukin-2 precursor is also available in UniProt accession number P60568 version 161. The amino acid sequence comprises a signal peptide spanning amino acids 1-20 of the interleukin-2 precursor and mature interleukin-2 spanning amino acids 21-153 of the precursor.

The nucleic acid sequence of the human interleukin-3 precursor mRNA is available at NCBI Accession No. NM_000586.3. Each amino acid sequence is available at NCBI Accession No. NP_000577.2.

The amino acid sequence of the human interleukin-3 precursor is also available in UniProt accession number P08700 version 177. The amino acid sequence comprises a signal peptide spanning amino acids 1-19 of the interleukin-3 precursor and mature interleukin 3 spanning amino acids 20-152 of the precursor.

Interleukin-4 (IL-4), preferably human interleukin-4 (hIL-4), is a multifunctional cytokine. Interleukin-4 is a ligand for the interleukin-4 receptor.

The nucleic acid sequence of the human interleukin-4 isoform 1 precursor mRNA is available at NCBI Accession No. NM_000589.3. The respective amino acid sequences of the interleukin-4 isoform 1 precursor are available at NCBI Accession No. NP_0005800.1.

The nucleic acid sequence of the human interleukin-4 isoform 2 precursor mRNA is available at NCBI Accession No. NM_172348.2. The respective amino acid sequences of the interleukin-4 isoform 2 precursor are available at NCBI Accession No. NP_758585.1.

The amino acid sequence of the human interleukin-4 precursor is also available in UniProt accession number P05112 version 178. The amino acid sequence comprises a signal peptide spanning amino acids 1 to 24 of the interleukin 4 isoform 1 and isoform 2 precursors.

Preferably, interleukin-4 comprises one or at least one of the amino acid sequences of SEQ ID NOs: 8-10, which are also illustrated in FIGS. 6a-6c, respectively.

The nucleic acid sequence of the human interleukin-5 precursor mRNA is available at NCBI Accession No. NM_000879.2. Each amino acid sequence is available at NCBI Accession No. NP_000870.1.

The amino acid sequence of the human interleukin-5 precursor is also available in UniProt accession number P05113 version 189. The amino acid sequence comprises a signal peptide spanning amino acids 1-19 of the interleukin-5 precursor and mature interleukin-5 spanning amino acids 20-134 of the precursor.

The nucleic acid sequence of the mRNA of the transcript variant 1 of the human interleukin-6 precursor is available at NCBI Accession No. NM_0000600.4. The respective amino acid sequences of the interleukin-6 isoform 1 precursor are available at NCBI Accession No. NP_000591.1.

The amino acid sequence of the human interleukin-6 precursor is also available in UniProt accession number P05231 version 212. The amino acid sequence comprises a signal peptide spanning amino acids 1-29 of the interleukin-6 precursor and a mature interleukin-6 spanning amino acids 30-212 of the precursor.

The nucleic acid sequence of the mRNA of the transcript variant 1 of the human interleukin-7 precursor is available at NCBI Accession No. NM_000880.3. The respective amino acid sequences of the interleukin-7 isoform 1 precursor are available at NCBI Accession No. NP_000871.1.

The amino acid sequence of the human interleukin-7 precursor is also available in UniProt accession number P05231 version 212. The amino acid sequence of the human interleukin-7 isoform 1 precursor comprises a signal peptide spanning amino acids 1 to 25 of the isoform 1 precursor and a mature interleukin-7 spanning amino acids 26 to 177 of the isoform 1 precursor. ..

The nucleic acid sequence of the mRNA of the transcript variant 1 of the human interleukin-8 precursor is available at NCBI Accession No. NM_0005844.3. The respective amino acid sequences of the interleukin-8 isoform 1 precursor are available at NCBI Accession No. NP_000575.1.

The amino acid sequence of the human interleukin-8 precursor is also available in UniProt accession number P10145 version 210. The amino acid sequence of the human interleukin-8 isoform 1 precursor comprises a signal peptide spanning amino acids 1-20 of the isoform 1 precursor. The 99 amino acid precursor peptide undergoes cleavage to produce several active IL8 isoforms.

Preferably, the mature human interleukin-8 comprises amino acids 31-99 of the interleukin-8 isoform 1 precursor.

Interleukin-9 (IL-9), preferably human interleukin-9 (hIL-9), is produced by a variety of cells. The nucleic acid sequence of the human interleukin-9 precursor mRNA can be found at NCBI Accession No. NM_0005900.1. Each amino acid sequence can be found in NCBI accession number NP_0005811.1 and UniProt accession number P15248 version 153.

The amino acid sequence of the human interleukin-9 precursor comprises a signal peptide spanning amino acids 1-18 of the human interleukin-9 precursor protein and a mature interleukin-9 spanning amino acids 19-144 of the precursor.

Interleukin-10 (IL-10), preferably human interleukin-10 (hIL-10), is a cytokine produced primarily by monocytes. The nucleic acid sequence of the human interleukin-10 precursor mRNA can be found at NCBI Accession No. NM_000572.2. Each amino acid sequence can be found in NCBI accession number NP_000563.1 and UniProt accession number P22301 version 156.

The amino acid sequence of the human interleukin-10 precursor comprises a signal peptide spanning amino acids 1-18 of the human interleukin-10 precursor protein.

Interleukin-12 is composed of two separate genes, the interleukin-12 subunit alpha (IL-12A), also referred to as p35, and the interleukin-12 subunit beta (IL-12B), also referred to as p40. It is a encoded heterodimer cytokine. An active heterodimer called "p70" and a homodimer of p40 are formed after protein synthesis.

The nucleic acid sequence of the human interleukin-12 subunit alpha precursor mRNA contains three transcript variants, NCBI Accession Nos. NM_0008822.4 (Variant 1), NM_0013545821 (Variant 2), and NM_0013545583. It is available in 1 (variant 3).

The amino acid sequences of the human interleukin-12 subunit alpha precursor isoforms 1, 2, and 3, respectively, are NCBI accession numbers NP_000873.2. Available at 170.

The human interleukin-12 subunit alpha isoform 1 precursor comprises a signal peptide spanning amino acids 1-56 of the precursor protein deposited in NCBI reference sequence: NP_000873.2.

Human IL12A variants 2 and 3 lack selective in-frame exons compared to variant 1. The resulting isoforms 2 and 3 have the same N- and C-termini but are shorter than isoform 1.

The nucleic acid sequence of the mRNA of the mouse interleukin-12 subunit alpha precursor contains two transcript variants, which are available at NCBI Accession Nos. NM_001159424.2 (Variant 1) and NM_0083351.3 (Variant 2). be.

The respective amino acid sequences of the mouse interleukin-12 subunit alpha precursor isoforms 1 and 2 are available in NCBI accession numbers NP_001152896.1 and NP_032377.1, and UniProt accession numbers P43431 version 140, respectively.

The mouse interleukin-12 subunit alpha isoform 2 precursor comprises a signal peptide spanning amino acids 1 to 22 of the precursor protein deposited in NCBI reference sequence: NP_032377.1.

Preferably, the interleukin-12 subunit alpha comprises the amino acid sequence of the mature mouse interleukin-12 subunit alpha isoform 2, which is illustrated in FIG. 13b and SEQ ID NO: 25.

The nucleic acid sequence of the human interleukin-12 subunit beta precursor mRNA is available at NCBI Accession No. NM_002187.2. Each amino acid sequence is available in NCBI accession number NP_002178.2. And UniProt accession number P29460 version 210.

The precursor of the human interleukin-12 subunit beta precursor comprises a signal peptide spanning amino acids 1 to 22 of the amino acid sequence deposited in NCBI reference sequence NP_002178.2.

The nucleic acid sequence of the mouse interleukin-12 subunit beta precursor mRNA is available at NCBI Accession No. NM_001303244.1. Each amino acid sequence is available in NCBI Accession No. NP_001290173.1 and UniProt Accession No. P43332 version 163.

The precursor of the mouse interleukin-12 subunit beta precursor comprises a signal peptide spanning amino acids 1 to 22 of the amino acid sequence deposited in NCBI reference sequence NP_001290173.1.

Preferably, the interleukin-12 subunit beta comprises the amino acid sequence of the mature mouse interleukin-12 subunit beta, which is illustrated in FIG. 13a and SEQ ID NO: 24.

More preferably, the interleukin-12 is a mature form of interleukin-12 subunit alpha and a mature form of interleukin-12 subunit beta, preferably comprising at least one amino acid linked by a linker sequence. It is expressed as a recombinant fusion protein containing.

More preferably, interleukin-12 is expressed as a recombinant fusion protein comprising one or at least one of the amino acid sequences of SEQ ID NOs: 26-27, which are also illustrated in FIGS. 13c and 13d, respectively.

Interleukin-13 (IL-13), preferably human interleukin-13 (hIL-13), is an immunoregulatory cytokine. The nucleic acid sequence of the human interleukin-13 precursor mRNA is available at NCBI Accession No. NM_002188.2. Each amino acid sequence is available in NCBI Accession No. NP_002179.2 and UniProt Accession No. P35225 Version 157.

The interleukin-13 precursor comprises a signal peptide spanning amino acids 1-24 of the interleukin-13 precursor protein deposited in UniProt accession number P35225 version 157.

Interleukin-18 (IL-18) is a cytokine belonging to the IL-1 superfamily and is produced, for example, by macrophages.

The nucleic acid sequence of the human interleukin-18 (hIL-18) precursor mRNA contains two transcript variants, which are utilized in NCBI accession numbers NM_001562.4 (variant 1) and NM_00124321.11 (variant 2). It is possible.

Transcript variant 1 corresponds to the major and longer transcript and encodes the longer hIL-18 isoform 1.

Transcript variant 2, also known as Delta3pro-IL-18, lacks in-frame code exons compared to variant 1. The encoded hIL-18 isoform 2 is shorter than the hIL-18 isoform 1 and may be resistant to proteolytic activation.

The respective amino acid sequences of human interleukin-18 precursor isoforms 1 and 2 are available in NCBI accession numbers NP_001553.1 and NP_001230140.1. And UniProt accession numbers Q14116 version 1174, respectively.

The human interleukin-18 isoform 1 precursor comprises a propeptide, which spans amino acids 1 to 36 of the precursor protein deposited in the NCBI reference sequence: NP_001553.1. And caspase 1 (CASP1) or caspase 4 in vivo. Processed by (Caspase 4) to produce an active form.

Preferably, interleukin-18 comprises one or at least one of the amino acid sequences of SEQ ID NOs: 16-18, which are also illustrated in FIGS. 10a-10c, respectively.

Interleukin-34 (IL-34) is a cytokine that also promotes monocyte and macrophage differentiation and viability. Due to alternative splicing, human interleukin-34 exists as two isoforms, which can be used in the present invention.

The nucleic acid sequence of the human interleukin-34 isoform 1 precursor mRNA is available at NCBI Accession No. NM_001172772.1. The corresponding amino acid sequence is available at NCBI Accession No. NP_0011662243.1.

The nucleic acid sequence of the human interleukin-34 isoform 2 precursor mRNA is available at NCBI Accession No. NM_00117277.1.1. The corresponding amino acid sequence is available at NCBI Accession No. NP_0011662242.1.

Each amino acid sequence is also available in UniProt accession number Q6ZMJ4 version 80. The human interleukin-34 precursor comprises a signal peptide spanning amino acids 1-20 of each amino acid sequence of the precursor protein.

The interleukin receptor antagonist protein is preferably selected from the interleukin-1 receptor antagonist protein (IL-1RA), the interleukin-36 receptor antagonist protein (IL-36RA), and mixtures thereof.

A suitable interleukin-binding protein is preferably interleukin-18-binding protein (IL-18BP).

The colony stimulating factor is preferably a polypeptide capable of stimulating proliferation, healing, and / or cell differentiation.

Preferably, the colony stimulating factor is colony stimulating factor 1 (CSF-1), colony-stimulating factor (GM-CSF), colony-stimulating factor (G-CSF), erythropoetin, thrombopoetin, and their functional analogs. , Their bio-stimulating factor, and their mixtures, preferably colony stimulating factor 1 (CSF-1), colony-stimulating factor (GM-CSF), colony-stimulating factor (G-CSF), erythropoetin, and the like. Selected from the group consisting of functional analogs of, their biosimilars, and mixtures thereof.

Colony-stimulating factor-1 (CSF-1), also known as macrophage colony-stimulating factor (M-CSF), is a cytokine that controls the production, differentiation, and function of macrophages.

Due to alternative splicing, human CSF-1 exists as different isoforms, which can be used in the present invention.

The nucleic acid sequence of human CSF-1 isoform 1 mRNA, also referred to as macrophage colony stimulating factor 1 isoform A precursor, is available at NCBI accession number NM_0007575. The corresponding amino acid sequence is available at NCBI Accession No. NP_000748.3.

The nucleic acid sequence of the mRNA of the human CSF-1 isoform 2 precursor, also referred to as the human macrophage colony stimulating factor 1 isoform B precursor, is available at NCBI Accession No. NM_172210.2. The corresponding amino acid sequence is available at NCBI Accession No. NP_757349.1.

The nucleic acid sequence of the mRNA of the human CSF-1 isoform 3 precursor, also referred to as the macrophage colony stimulating factor 1 isoform C precursor, is available at NCBI Accession No. NM_172211.3. The corresponding amino acid sequence is available at NCBI Accession No. NP_757350.1.

Each amino acid sequence is also available in UniProt accession number P09603 version 158. Isoform 1 was chosen as the orthodox UniProt sequence.

Each protein sequence of human CSF-1 precursor isoforms 1 to 3 comprises an N-terminal signal peptide spanning amino acid numbers 1 to 32 of each amino acid sequence.

The active form of human CSF-1 can be found extracellularly as a disulfide-linked homodimer. The active form is produced by proteolytic cleavage of the membrane-bound precursor, resulting in the loss of the N-terminal signal peptide.

Preferably, colony stimulating factor 1 comprises one or at least one of the amino acid sequences of SEQ ID NOs: 11-13, which are also illustrated in FIGS. 7a-7c, respectively.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is also known as colony stimulating factor 2 (CSF-2).

The nucleic acid sequence of the human granulocyte-macrophage colony-stimulating factor precursor mRNA is available at NCBI Accession No. NM_000758.3. Each amino acid sequence of the GM-CSF precursor is available at NCBI Accession No. NP_000749.2.

The amino acid sequence of the human GM-CSF precursor is also available in UniProt accession number P04141 version 180. The amino acid sequence of the human GM-CSF precursor comprises a signal peptide spanning amino acids 1 to 17 of the precursor and a mature GM-CSF straddling amino acids 18 to 144 of the GM-CSF precursor.

Preferably, the granulocyte-macrophage colony stimulating factor (GM-CSF) comprises one or at least one of the amino acid sequences of SEQ ID NOs: 19-21, which are also illustrated in FIGS. 11a-11c, respectively.

Granulocyte colony stimulating factor (G-CSF or GCSF) is also known as colony stimulating factor 3 (CSF3).

In humans, preferably two different G-CSF polypeptides, called isoform 1 and isoform 2, are synthesized from the same gene by differential splicing of mRNA. The two polypeptides differ only in the presence or absence of the three amino acids. Expression studies indicate that both preferably have G-CSF activity.

The nucleic acid sequence of the mRNA of the human G-CSF isoform 1 precursor, also referred to as the G-CSF isoform a precursor, is available at NCBI Accession No. NM_000759.3. The corresponding amino acid sequence is available at NCBI Accession No. NP_000750.1.

The nucleic acid sequence of the mRNA of the human G-CSF isoform 2 precursor, also referred to as the G-CSF isoform b precursor, is available at NCBI Accession No. NM_172219.2. The corresponding amino acid sequence is available at NCBI Accession No. NP_757373.1.

Each amino acid sequence of human G-CSF is also available in UniProt accession number P09919 version 193. Isoform 1 was chosen as the orthodox UniProt sequence.

Each protein sequence of human G-CSF precursor isoforms 1 and 2 comprises an N-terminal signal peptide spanning amino acid numbers 1 to 29 of the respective amino acid sequence.

Erythropoietin (EPO) stimulates the production of red blood cells (erythropoiesis) in the bone marrow.

The nucleic acid sequence of the human erythropoietin precursor mRNA is available at NCBI Accession No. NM_000799.3. Each amino acid sequence of the erythropoietin precursor is available at NCBI Accession No. NP_000790.2.

The amino acid sequence of the human erythropoietin precursor is also available in UniProt accession number P01588 version 183. The amino acid sequence of the human erythropoietin precursor comprises a signal peptide spanning amino acids 1-27 of the precursor and mature erythropoietin spanning amino acids 28-193 of the precursor.

Chemokines are preferably a group of chemotactic polypeptides that control the recruitment of various leukocytes to the site of inflammation. Chemokines can be subdivided into C, CC _, CXC, and CX3C subgroups based on the homology of each polypeptide sequence and the number of amino acids between the first two cysteines.

Preferably, chemokines are selected from the group consisting of CCL1 to CCL28, CXCL1 to CXCL17, XCL1, XCL2, CX3CL1, their functional analogs, their biosimilars, and mixtures thereof.

Examples of polypeptide hormones are known to those of skill in the art and include, for example, corticotropin-releasing hormone, pancreatic islet amyloid polypeptide, gastric inhibitory polypeptide, grelin, glucagon-like peptide-1, growth hormone-releasing hormone, appetite-regulating hormone, insulin, leptin, etc. Motilin, thyrotropin-releasing hormone, urocortin, pancreatic polypeptide, somatostatin, sodium diuretic peptide, growth hormone, prolactin, calcitonin, luteinizing hormone, parathyroid hormone, thyroid stimulating hormone, vasoactive intestinal peptide, their functional analogs, Includes their biosimilars, and mixtures thereof.

Examples of neuropeptides are known to those of skill in the art and include, for example, Aguthi-related proteins (AgRP), gastrin-releasing peptides (GRP), calcitonin gene-related peptides (CGRP), cocaine and amphetamine-regulated transcript peptides (CART), dynorfins, etc. Endolphin, enterostatin, galanine peptide (GAL), galanin-like peptide (GALP), hypocretin / olexin, melanin aggregation hormone (MCH), neuromedin, neuropeptide B, neuropeptide K, neuropeptide S, neuropeptide W, neuropeptide Y , Neurotensin, Oxytocin, Prolactin-releasing peptide, Proopiomelanocortin and its derived melanocortin, Apolypoprotein A-IV, Oxintmodulin, Gastrin-releasing peptide, Glucose-dependent insulin secretion-stimulating polypeptide, Orphanin FQ, Enkephalin, their functions Includes peptides, their biosimilars, and mixtures thereof.

Examples of enzymes are known to those of skill in the art and include, for example, eukaryotic thrombolytic enzymes, including tissue plasminogen activators, urokinases, or other enzymes such as Factor VII activating proteases.

The antibodies described herein can be full-sized antibodies, or functional fragments thereof, such as Fabs, fusion proteins, or multimeric proteins.

As used herein, the term "functional" refers to an antibody fragment that is still capable of exercising its intended function, namely antigen binding. The term antibody used here refers to conventional antibody, chimeric antibody, dAb, bispecific antibody, trispecific antibody, multispecific antibody, bivalent antibody, trivalent antibody, multivalent antibody, VHH, nanobody. , Fab, Fab', F (ab') 2, scFv, Fv, dAb, Fd, diabody, triabody, single-stranded antibody, single domain antibody, single antibody variable domain, but limited to these. Not done.

In this context, the term "antibody" is used to describe immunoglobulins, whether natural, partially, or wholly manipulated. Since antibodies can be modified in a number of ways, the term "antibody" refers to antibody fragments, derivatives, functional equivalents, and homologs of antibodies, as well as single-chain antibodies, bifunctional antibodies, bivalent antibodies, VHHs, nanobodies. , Fab, Fab', F (ab') 2, scFv, Fv, dAb, Fd, diabody, triabody, and camel antibodies, covered with natural, whole, or partially manipulated. Includes any polypeptide containing an immunoglobulin binding domain, whether or not. Thus, a chimeric molecule comprising an immunoglobulin binding domain or equivalent fused to another polypeptide is included. The term also covers any polypeptide or protein that has a binding domain that is, or is homologous to, an antibody binding domain, such as an antibody mimic.

Examples of antibodies are immunoglobulin isotypes and their isotype subclasses, including IgG (IgG1, IgG2a, IgG2b, IgG3, IgG4), IgA, IgD, IgM, and IgE. Therefore, one of ordinary skill in the art will appreciate that the present invention also relates to antibody fragments containing antigen binding domains such as VHH, Nanobodies, Fabs, scFv, Fv, dAb, Fd, diabody and triabody. Let's do it. In certain embodiments, the invention relates to a gram-positive bacterium or recombinant nucleic acid described herein, wherein one exogenous gene encodes a light chain (VL) of an antibody or functional fragment thereof and another. The exogenous gene encodes a heavy chain (VH) of an antibody or functional fragment thereof, more preferably the functional fragment is Fab. In certain embodiments, the exogenous gene encoding VL or a functional fragment thereof is transcriptionally coupled to the 3'end of the exogenous gene encoding VH or a functional fragment thereof.

In a preferred embodiment, the recombinant bacterium of the invention expresses 1, 2, 3, 4, or more heterologous factors, each of which is not naturally present in or expressed by the bacterium used. Instead, independently, it is a heterologous polypeptide or complex thereof, preferably a heterologous polypeptide or complex thereof.

For example, if the recombinant bacterium of the invention expresses 2, 3, 4, or more heterologous factors, each expression of each heterologous factor is controlled by the chloride-inducible promoters of the individual prokaryotes. Or even more preferably, it can be controlled by a single prokaryotic chloride-inducible promoter that controls the expression of heterologous factors from a single operon.

For example, each nucleic acid sequence encoding each heterologous factor may be located in a separate subpopulation of the recombinant bacterium, which may be combined to give the recombinant bacterium of the invention.

Each individual subpopulation containing each nucleic acid sequence can be, for example, from the same or different species of bacteria.

For example, different species of recombinant bacteria expressing different heterologous factors can be used to adapt the expression and preferably release of each heterologous factor from the recombinant bacterium.

In a preferred embodiment of the invention, each nucleic acid sequence (s) encoding at least one heterologous factor is present in the recombinant bacterium in varying numbers of copies. For example, recombinant bacteria contain at least one copy of each nucleic acid sequence encoding at least one heterologous factor per bacterial cell.

In order to enhance the expression of each heterologous factor, the copy number of each nucleic acid sequence present in each bacterial cell, preferably in the recombinant bacterium, can be increased independently.

For example, if two or more heterologous factors should be expressed by the recombinant bacterium, different ratios of copies of each nucleic acid sequence encoding the heterologous factor may be present in the bacterial cell of the recombinant bacterium. ..

In a preferred embodiment, the nucleic acid sequence encoding at least one, preferably one, two, three, four, or more heterologous factors (s) is located in one population of recombinant bacteria.

In a more preferred embodiment, each nucleic acid sequence (s) encoding at least one heterologous factor resides on at least one of the recombinant bacterial chromosomes and plasmids.

Placing a nucleic acid sequence on a recombinant bacterial chromosome or on a recombinant bacterial plasmid determines, among other things, the amount of protein translated by the bacterium, because the level of expression from the plasmid is the level of expression from the chromosome. This is because it is expensive compared to.

In a more preferred embodiment of the invention, at least one nucleic acid sequence encoding each heterologous factor is provided on the bacterial chromosome and at least one nucleic acid sequence encoding another heterologous factor is provided on the recombinant bacterial plasmid. Will be done.

In an alternative embodiment of the invention, each nucleic acid sequence is provided either on an individual portion of the recombinant bacterium's chromosome or on a different plasmid of the recombinant bacterium.

In a preferred embodiment of the invention, all nucleic acid sequences encoding at least one, preferably 1, 2, 3, 4, or more heterologous factors (s) are single recombinant nucleic acids, preferably. Provided on a recombinant plasmid.

More preferably, the recombinant nucleic acids of the invention, preferably recombinant plasmids, contain 1, 2, 3, 4, or more nucleic acid sequences encoding 1, 2, 3, 4, or more factors, respectively. Each of these is not naturally present in or expressed by the bacterium used, and these are heterologous polypeptides or complexes thereof.

In another particularly preferred embodiment of the invention, at least one nucleic acid sequence encoding at least one, preferably one, two, three, four, or more heterologous factors (s) is a single operon. Located above, it is operably linked to and controlled by a chloride-inducible promoter of a single prokaryotic system, the activity of which is controlled by at least one prokaryotic regulator gene.

More preferably, the chloride-inducible promoter and at least one regulator gene are located on the chloride-inducible gene expression cassette, whereas the chloride-inducible promoter is located downstream from at least one regulator gene. The chloride-induced gene expression cassette controls transcription of at least one nucleic acid sequence encoding at least one heterologous factor, more preferably located on a single operon.

An operon is a functional unit of DNA that contains a cluster of genes under the control of a single promoter. Genes are transcribed together from the mRNA strand and translated together in the cytoplasm.

Preferably, each nucleic acid sequence encoding each heterologous factor located on a single operon is provided with a nucleic acid sequence encoding a ribosome binding site for translation initiation.

Preferably, the nucleic acid sequence encoding the ribosome binding site is located upstream of the start codon, which is the direction of the 5'region of the same strand to be transcribed. The sequence is preferably complementary to the 3'end of 16S ribosomal RNA.

Preferably, each nucleic acid sequence encoding each heterologous factor located on a single operon is provided with a nucleic acid sequence encoding a secretory signal sequence at the 5'end of the open reading frame (ORF) of the heterologous factor. It produces a fusion protein containing a secretory signal sequence and a heterologous factor.

In a preferred embodiment of the recombinant bacterium of the invention or the recombinant plasmid of the invention, the chloride-inducible promoter and / or at least one regulator gene are each independently from the same bacterial species.

In a preferred embodiment of the invention, the chloride-inducible promoter is a taxonomic species of the order Lactobacillus, preferably PgadC from Lactococcus lactis.

In a preferred embodiment of the invention, the regulatory factor gene encodes GadR from a taxonomic species of the order Lactococcus lactis, preferably Lactococcus lactis, preferably available at GenBank Accession No. AAC46186.1. Encodes a polypeptide containing an amino acid sequence.

More preferably, even more preferably, the expression of the regulator gene, which encodes GadR from a taxonomic species of the order Lactobacillus, preferably Lactococcus lactis, is regulated by a constitutive promoter.

Preferably, a nucleic acid sequence encoding the positive regulators GadR (gadR), GadC (gadC), and glutamate decarboxylase (gadB) of Lactococcus lactis is available at GenBank Accession No. AAC46187.1.

In a preferred embodiment of the invention, the chloride-induced gene expression cassette comprises or consists of the nucleic acid sequence of SEQ ID NO: 2, also illustrated in FIG. 2b, and encodes at least one heterologous factor. It controls the transcription of one nucleic acid sequence, which is more preferably located on a single operon.

Preferably, the recombinant bacterium of the invention is a prokaryotic chloride-inducible bacterium, preferably selected from the taxonomic phylum filmites, the taxonomic phylum actinobacteria, or the taxonomic phylum proteobacterium. At least one nucleic acid sequence that is functionally coupled to a promoter and encodes at least one heterologous factor, the heterologous factor is independently a heterologous polypeptide or complex thereof, and the heterologous polypeptide is at least one. It is obtained by transformation with at least one prokaryotic regulator gene comprising a eukaryotic polypeptide, at least one fragment thereof, or a combination thereof and controlling the activity of a chloride-inducible promoter.

Proteobacteria are the major phyla of Gram-negative bacteria, including a wide variety of known bacterial genera, such as Escherichia, Salmonella, Vibrio, Helicobacter, Yersinia, Legionellales.

Suitable bacteria for obtaining the recombinant bacterium of the present invention are preferably non-pathogenic bacteria. Preferably, the non-pathogenic bacterium is a non-invasive bacterium.

In a more preferred embodiment, the recombinant bacterium is a Gram-positive bacterium, preferably a Gram-positive non-spore-forming bacterium. More preferably, the bacterium is a non-establishing bacterium that lacks the ability to colonize the human gastrointestinal tract.

In a more preferred embodiment, the recombinant bacterium comprises a Gram-positive food grade bacterium strain, preferably a facultative anaerobic bacterium strain.

According to a preferred embodiment of the invention, the recombinant bacterium can be from the same bacterial strain or a mixture of different bacterial strains.

In another embodiment, the bacterium is classified by the US Food and Drug Administration (FDA) as "Generally Recognized As Safe" (GRAS).

In another embodiment, the bacterium has a "safety eligibility estimate" (QPS status) as defined by the European Food Safety Authority (EFSA). The introduction of a safety eligibility estimation (QPS) approach for the evaluation of selected microorganisms is described in EFSA Journal, Vol. 587,2007, pages 1-16.

In a more preferred embodiment, the bacterium is a non-pathogenic bacterium belonging to the taxonomic phylum Bacillota or Actinobacteria. Preferably, the bacterium is from at least one genus selected from the group consisting of Bifidobacterium, Corinebacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Propionibacterium, and Streptococcus. It is a non-pathogenic bacterium.

In another preferred embodiment, the recombinant bacterium is a lactic acid bacterium (LAB). Lactic acid bacteria are a gram-positive, acid-tolerant, generally non-spore-forming, non-respiratory bacterial clade that shares common metabolic and physiological characteristics. These bacteria produce lactic acid as the major metabolic end product of carbohydrate degradation.

Lactic acid bacteria are known and are used, for example, in food fermentation. Moreover, proteinaceous bacteriocins are produced by several lactic acid bacterial strains.

In a more preferred embodiment, the bacteria used to obtain the recombinant bacteria of the invention exclude pathogenic and / or opportunistic bacteria.

In another embodiment, the bacterium is the genus Bifidobacterium sp. From Bifidobacterium actinocoloniform, Bifidobacterium addresscentis, Bifidobacterium asclepiy, Bifidobacterium angularum, Bifidobacterium, Bifidobacterium animalis, for example Bi Fidobacterium animalis subspecies Animalis or Bifidobacterium animalis subspecies Lactis, Bifidobacterium asteroides, Bifidobacterium biavati, Bifidobacterium bifidam, Bifidobacterium bohemicum, Bifidobacterium Um Bombi, Bifidobacterium Boum, Bifidobacterium Breve, Bifidobacterium Calitricos, Bifidobacterium Catenuratam, Bifidobacterium Kerinum, Bifidobacterium Corineform, Bifidobacterium Um Kurdilactis, Bifidobacterium kunikuri, Bifidobacterium denticorence, Bifidobacterium dentium, Bifidobacterium faecale, Bifidobacterium gallicum, Bifidobacterium galinalum, Bifidobacterium globosamm, Bifidobacterium indicum, Bifidobacterium infantis, Bifidobacterium inopinatsum, Bifidobacterium kashiwanohens, Bifidobacterium lactis, Bifidobacterium Longum, such as Bifidobacterium longum subspecies Infantis, Bifidobacterium longum subspecies Longum, or Bifidobacterium longum subspecies Switzerland, Bifidobacterium magnum, Bifidobacterium melisicum, Bifidobacterium minimum, Bifidobacterium mongolians, Bifidobacterium moucarabens, Bifidobacterium pseudocatenuratam, Bifidobacterium pseudolongum, for example Bifidobacterium pseudorongam Subspecies Globosam or Bifidobacterium pseudorongam Subspecies Pseudoron gum, Bifidobacterium cyclellophyllum, Bifidobacterium prolam, Bifidobacterium reuteri, Bifidobacterium luminantium, Bifidobacterium seculare, Bifidobacterium saguini, Bifidobacterium scaldobii, Bifidobacterium Mu Stelenbochens, Bifidobacterium subtilis, Bifidobacterium stercolis, Bifidobacterium Switzerland, Bifidobacterium thermacidophilum, eg Bifidobacterium thermacidophilum subspecies Includes, but is not limited to, porcinum or bifidobacteria thermacidophilum subspecies thermacidophilum, bifidobacteria thermophyllum, or bifidobacteria turmiens.

Preferably, the bacterium is not Bifidobacterium dentium.

Preferably, the bacterium is the genus Bifidobacterium sp. Bacteria with "Safety Eligibility Estimate" (QPS) status, Bifidobacterium addresscentis, Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium. Includes, but is not limited to, Umm Bifidum.

In one embodiment, the bacterium is the genus Corynebacterium sp. From Corynebacterium acorence, Corynebacterium afermentans, such as Corynebacterium afermentans subspecies Afermentans or Corynebacterium afermentans subspecies Lipophyllum, Corynebacterium ammoniagenes. , Corynebacterium amicolatum, Corynebacterium appendices, Corynebacterium aquatimens, Corynebacterium aquille, Corynebacterium algentraten, Corynebacterium atipicum, Corynebacterium aurimcosam, Corynebacterium -Auris, Corynebacterium auris canis, Corynebacterium betae, Corynebacterium beticola, Corynebacterium bovis, Corynebacterium carnae, Corynebacterium campoleensis, Corynebacterium canis, Corynebacterium Um Capitobis, Corynebacterium casei, Corynebacterium caspium, Corynebacterium siconiae, Corynebacterium confsum, Corynebacterium koileae, Corynebacterium cistidis, Corynebacterium deserti, Coryne Corynebacterium diphtheniae, Corynebacterium dosanens, Corynebacterium durum, Corynebacterium efficiens, Corynebacterium epidermidicanis, Corynebacterium equi, Corynebacterium falsenii, Corynebacterium fascianus , Corynebacterium ferrinum, Corynebacterium fracum faciens, such as Corynebacterium fracum faciens subspecies Betae, Corynebacterium fracum faciens subspecies Fracum faciens, Corynebacterium fracum Faciens Subspecies Allty, or Corynebacterium Fracum Faciens Subspecies Poinsetiae, Corynebacterium Flabescens, Corynebacterium Frankenforstens, Corynebacterium Freiburgens, Corynebacterium Freney, Coryne Corynebacterium glaucom, Corynebacterium glucuronoricam, Corynebacterium glutamicum, Corynebacterium halotolerance, Corynebacterium Hansenii, Corynebacterium hoagii, Corynebacterium humirejuse Corynebacterium irisis, Corynebacterium imitans, Corynebacterium insidiosum, Corynebacterium iranicum, Corynebacterium J-caium, Corynebacterium croppenstetti, Corynebacterium kutsheri, Coryne Corynebacterium lactis, Corynebacterium lylium, Corynebacterium lipophiloflavum, Corynebacterium liqufaciens, Corynebacterium rubricantis, Corynebacterium McGinley, Corynebacterium marinum, Corynebacterium Umm Maris, Corynebacterium maciliens, Corynebacterium mastitidis, Corynebacterium matrutii, Corynebacterium miciganens, such as Corynebacterium miciganens subspecies Insidiosum, Corynebacterium miciganens subspecies Michiganens , Corynebacterium Michiganens subspecies Nebraskens, Corynebacterium Michiganens subspecies Sepedonicum, or Corynebacterium Michiganens subspecies Tecerarius, Corynebacterium minutissimam, Corynebacterium Mooreparkens, Corynebacterium musifaciens, Corynebacterium Corynebacterium Mustellae, Corynebacterium Mycetoides, Corynebacterium Nebraskens, Corynebacterium Niglycans, Corynebacterium Nuruki, Corynebacterium Alti, Corynebacterium Paulo Metabolum, Corynebacterium Fokae, Coryne Corynebacterium Pyrvalence, Corynebacterium Pyrosam, Corynebacterium Poinsettiae, Corynebacterium Propinkum, Corynebacterium Pseudogiftericum, Corynebacterium Pseudotubercrosis, Corynebacterium pyogenes, Corynebacterium pyrubiciproduces, Corynebacterium lasai, Corynebacterium renale, Corynebacterium registence, Corynebacterium riegerii, Corynebacterium seminale, Corynebacterium sepedonicum, Corynebacterium sim. Reims, Corynebacterium Singulare, Corynebacterium spenice, Corynebacterium spenicecolm, Corynebacterium spouty, Corynebacterium statusoni Corynebacterium striatum, Corynebacterium suicordis, Corynebacterium sundosvalence, Corynebacterium turpenotavidum, Corynebacterium testudinoris, Corynebacterium somsenii, Corynebacterium thymonens, Corynebacterium. Tritisi, Corynebacterium tubercrostalicum, Corynebacterium tuscaniens, Corynebacterium urselanth, Corynebacterium urseribobis, Corynebacterium urealicum, Corynebacterium ureiseleribolance, Corynebacterium • Includes, but is not limited to, Utereki, Corynebacterium barrierville, Corynebacterium Vitaerminis, or Corynebacterium zerosis.

Preferably, the bacteria are Corynebacterium diphtheria, Corynebacterium amicolatum, Corynebacterium striatum, Corynebacterium J-caium, Corynebacterium urealicum, Corynebacterium zerosis, Corynebacterium. Not Pseudotubercrosis, Corynebacterium tenuis, Corynebacterium striatum, or Corynebacterium minutisimum.

Preferably, the bacterium is a bacterium classified as "generally recognized safe" (GRAS) of the genus Corynebacterium, Corynebacterium ammoniagenes, Corynebacterium casei, Corynebacterium flavesens. , Or include, but is not limited to, the Corynebacterium barrier building.

In another embodiment, the bacterium is the genus Enterococcus sp. Enterococcus arcedinis, Enterococcus aquimarinus, Enterococcus asini, Enterococcus abium, Enterococcus kakae, Enterococcus camelliae, Enterococcus canintestini, Enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus. Enterococcus diestramnae, Enterococcus Disper, Enterococcus durance, Enterococcus Eurekensis, Enterococcus faecalis, Enterococcus Fesium, Enterococcus flavesens, Enterococcus enterococcus galinarum, Enterococcus enterococcus Enterococcus Italix, Enterococcus lactis, Enterococcus remanii, Enterococcus Marodolatus, Enterococcus morabiensis, Enterococcus Munchii, Enterococcus olivae, Enterococcus parence, Enterococcus enterococcus Enterococcus, Enterococcus kevesensis, Enterococcus raffinosas, Enterococcus ratti, Enterococcus ribolam, Enterococcus rotai, Enterococcus saccharoriticus, such as Enterococcus saccharoriticus subspecies Saccharolitas or Enterococcus saccharoticus. Enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococcus enterococci Includes, but is not limited to, enterococcus.

Preferably, the bacterium is a bacterium classified as "generally recognized safe" (GRAS) of the genus Enterococcus, including, but limited to, Enterococcus durance, Enterococcus faecalis, or Enterococcus faecium. Not done.

In another embodiment, the bacterium is the genus Lactobacillus sp. Lactobacillus acetotolerance, Lactobacillus acidifalinae, Lactobacillus acidipistis, Lactobacillus acidophilus, Lactobacillus aguillis, Lactobacillus argidas, Lactobacillus arimentalius, Lactobacillus amyloriticas, Lactobacillus aminophilus Lactobacillus, Lactobacillus amyloboras, Lactobacillus animalis, Lactobacillus antri, Lactobacillus apinorm, Lactobacillus apis, Lactobacillus apodemi, Lactobacillus aquaticus, Lactobacillus arisonensis, Lactobacillus arabalis, for example Lactobacillus subspecies Abianius, Lactobacillus bacchy, Lactobacillus bavarix, Lactobacillus bifermentans, Lactobacillus bovalius, Lactobacillus bombi, Lactobacillus blantae, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus bulgaricus Lactobacillus cameliae, Lactobacillus capillatus, Lactobacillus carnis, Lactobacillus casei, such as Lactobacillus casei subspecies Lactobacillus casei subspecies Casei, Lactobacillus casei subspecies Pseudoplantalum, Lactobacillus casei subspecies Lactobacillus casei Casei Subspecies Tolerance, Lactobacillus catenaformis, Lactobacillus cellobiosas, Lactobacillus seti, Lactobacillus choreohominis, Lactobacillus corinoides, Lactobacillus composti, Lactobacillus concabus, Lactobacillus confusas, Lactobacillus coriniformis, for example Formis subspecies Coriniformis or Lactobacillus Coriniformis subspecies Torkens, Lactobacillus Chrisspatus, Lactobacillus crustrum, Lactobacillus currierae, Lactobacillus carbatas, such as Lactobacillus carbatas subspecies Lactobacillus carbatas subspecies Melibiosus, Lactobacillus・ Shiprikazei, Lactobacillus delbrecchii, for example, Lactobacillus delbres Lactobacillus bulgaricas, Lactobacillus delbreckii subspecies Delbreckii, Lactobacillus delbrecchii subspecies Indicas, Lactobacillus delbrecchii subspecies Jacobseni, Lactobacillus delbrecchii subspecies Lactis, or Lactobacillus delbrecchii subspecies Sunky, Lactobacillus de Lactobacillus de Lactobacillus Lactobacillus divergens, Lactobacillus durianis, Lactobacillus equis, Lactobacillus eccasolis, Lactobacillus equigenerosi, Lactobacillus fabifermentans, Lactobacillus faesis, Lactobacillus falciminis, Lactobacillus faraginis, Lactobacillus faraginis Lactobacillus fermentum, Lactobacillus floricola, Lactobacillus floram, Lactobacillus fornicalis, Lactobacillus fluctibolans, Lactobacillus fructosus, Lactobacillus flumenti, Lactobacillus fuchuensis, Lactobacillus flufricola, Lactobacillus fuza Lactobacillus gastricus, Lactobacillus ganensis, Lactobacillus gigarioram, Lactobacillus graminis, Lactobacillus halotrerance, Lactobacillus hamesii, Lactobacillus hamsteri, Lactobacillus harbinensis, Lactobacillus harbinensis, Lactobacillus harbinensis, Lactobacillus hayakitensis Lactobacillus helsylluensis, Lactobacillus helveticas, Lactobacillus heterohiotis, Lactobacillus hirugardi, Lactobacillus hokkaidonensis, Lactobacillus hominis, Lactobacillus homohiotis, Lactobacillus holdi, Lactobacillus inrs Lactobacillus gensenii, Lactobacillus Johnsonii, Lactobacillus Calisensis, Lactobacillus candolelli, Lactobacillus kefilanofaciens, such as Lactobacillus kefilanofaciens subspecies Kefilanofaciens or Lactobacillus kefilanofaciens Lactobacillus Lactobacillus, Lactobacillus kefilgranum, Lactobacillus kimbradii, Lactobacillus kimchix, Lactobacillus kimtiensis, Lactobacillus kimchii, Lactobacillus xsonensis, Lactobacillus kitasasatnis, Lactobacillus coriensis, Lactobacillus claliberi Lactobacillus Lakemannii, Lactobacillus Lindneri, Lactobacillus malefermentans, Lactobacillus mari, Lactobacillus maltalomics, Lactobacillus manifestibolans, Lactobacillus meliffer, Lactobacillus melis, Lactobacillus melibentris, Lactobacillus meliben , Lactobacillus minutas, Lactobacillus mucosae, Lactobacillus murinas, Lactobacillus nagerii, Lactobacillus Namrensis, Lactobacillus Nantensis, Lactobacillus nasensis, Lactobacillus Nenjangensis, Lactobacillus nodensis, Lactobacillus odoratif Oligofermentans, Lactobacillus orris, Lactobacillus oryzei, Lactobacillus otakuensis, Lactobacillus ozensis, Lactobacillus panis, Lactobacillus pancelis, Lactobacillus parabrevis, Lactobacillus parabufuneri, Lactobacillus paracasei, for example Lactobacillus paracasei Lactobacillus subspecies tolerance, Lactobacillus paracorinoides, Lactobacillus parafalaginis, Lactobacillus parakefiris, Lactobacillus paralimentalius, Lactobacillus paraplantalm, Lactobacillus pasteurii, Lactobacillus pausiborans, Lactobacillus pausiborans, Lactobacillus pentossus Lactobacillus plantalum, such as Lactobacillus plantalum subspecies Argentratensis or Lactobacillus plantalum subspecies Plantalum, Lactobacillus pobjii, Lactobacillus pontis, Lactobacillus porcinae, Lactobacillus sittasi, Lactobacillus lapis, Lactobacillus renni Las Reuteri, Lactobacillus ramnosus, Lactobacillus limae, Lactobacillus rhodentium, Lactobacillus logosae, Lactobacillus Russiae, Lactobacillus luminis, Lactobacillus saerimunelli, Lactobacillus sakei, such as Lactobacillus sakei subspecies Carnosus or Lactobacillus Species Lactobacillus salivarius, such as Lactobacillus salivarius subspecies Lactobacillus or Lactobacillus salivalius subspecies Lactobacillus sanfrancisensis, Lactobacillus sanibili, Lactobacillus satumensis, Lactobacillus secariphilus, Lactobacillus Lactobacillus spicerry, Lactobacillus spicerry, Lactobacillus spicerry, Lactobacillus spicerry, Lactobacillus spicerry , Lactobacillus suevicus, Lactobacillus Sunkii, Lactobacillus Santrieus, Lactobacillus Thaiwanensis, Lactobacillus tylandensis, Lactobacillus Thermotrerance, Lactobacillus trichodes, Lactobacillus Tossetti, Lactobacillus lili, Lactobacillus alchus Lactobacillus stelcus, Lactobacillus baginaris, Lactobacillus bersmordensis, Lactobacillus vini, Lactobacillus bilidecence, Lactobacillus bitulinus, Lactobacillus champungensis, Lactobacillus xylosas, Lactobacillus yamanasiensis, for example. Mali, or the subspecies Lactobacillus Yamanasiensis, including, but not limited to, Lactobacillus yonginensis, Lactobacillus diae, or Lactobacillus zimae.

Preferably, the bacterium is the genus Lactobacillus sp. Lactobacillus acidophilus strain NP28, Lactobacillus acidophilus strain NP51, Lactobacillus subspecies Lactis strain NP7, Lactobacillus reuteri strain NCIMB30242, Lactobacillus casei Lactobacillus reuteri strain DSM17938, Lactobacillus reuteri strain NCIMB30242, Lactobacillus acidophilus strain NCFM, Lactobacillus ramnosus strain HN001, Lactobacillus ramnosus strain HN001, Lactobacillus subsylus G Lactobacillus, Lactobacillus lactis, Lactobacillus acetotolerance, Lactobacillus acidifalinae, Lactobacillus acidipistis, Lactobacillus acidophilus, Lactobacillus arimentalius, Lactobacillus amyloricus, Lactobacillus amyloboras, Lactobacillus brevis Lactobacillus casei subspecies Casei, Lactobacillus corinoides, Lactobacillus composti, Lactobacillus coriniformis subspecies Coriniformis, Lactobacillus crispatus, Lactobacillus crustrum, Lactobacillus carbatas subspecies Lactobacillus delbrecchii Species Lactobacillus, Lactobacillus delbrecchii subspecies Delbrecchii, Lactobacillus delbrecchii subspecies Lactis, Lactobacillus dextrinicus, Lactobacillus diolibolans, Lactobacillus favifermentans, Lactobacillus farsiminis, Lactobacillus fermentum, Lactobacillus fluc , Lactobacillus flumenti, Lactobacillus gasseri, Lactobacillus ganensis, Lactobacillus hamesii, Lactobacillus harbinensis, Lactobacillus helveticas, Lactobacillus hirugardi, Lactobacillus homohiochii, Lactobacillus holdi, Lactobacillus gensevi・ Kefiranofadens subspecies Kefiranofaciens, Lactobacillus kefilanofadens subspecies Lactobacillus, Lactobacillus kimchii, Lactobacillus xsonensis, Lactobacillus mail, Lactobacillus manihotivolance, Lactobacillus mindensis, Lactobacillus mucosae, Lactobacillus nuggeri, Lactobacillus Namrensis, Lactobacillus nantensis, Lactobacillus nantensis, Lactobacillus nantensis Otakiensis, Lactobacillus Panis, Lactobacillus parabrevis, Lactobacillus parabufunelli, Lactobacillus paracasei subspecies Paracasei, Lactobacillus parakephili, Lactobacillus paralymentalius, Lactobacillus paraplantalm, Lactobacillus pentosus, Lactobacillus Plantalum, Lactobacillus pobjii, Lactobacillus pontis, Lactobacillus lapis, Lactobacillus reuteri, Lactobacillus ramnosus, Lactobacillus Russiae, Lactobacillus sakei subspecies Carnosas, Lactobacillus sakei subspecies Sakei, Lactobacillus salibalis subspecies Sarivarius Sensis, Lactobacillus satumensis, Lactobacillus secariphyrus, Lactobacillus senmaiskei, Lactobacillus silliginis, Lactobacillus spicerry, Lactobacillus suevicas, Lactobacillus sunkii, Lactobacillus tossetti, Lactobacillus vactinos -Includes, but is not limited to, Yamanasiensis.

Preferably, the bacterium is the genus Lactobacillus sp. Lactobacillus acidophilus, Lactobacillus amyloricus, Lactobacillus amylovoras, Lactobacillus Alimentarius, Lactobacillus abiaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus・ Casei, Lactobacillus crispatus, Lactobacillus carbatas, Lactobacillus delbrecchii, Lactobacillus falsimilis, Lactobacillus fermentum, Lactobacillus galinalum, Lactobacillus gasseri, Lactobacillus helveticas, Lactobacillus hirugardi, Lactobacillus syllabus Faciens, Lactobacillus kefili, Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus paraplantalm, Lactobacillus pentosas, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus ramnosus • Includes, but is not limited to, Sullivarius, Lactobacillus sanfrancisensis, or Lactobacillus diae.

In another embodiment, the bacterium is the genus Lactococcus sp. Lactococcus chungangensis, Lactococcus formocensis, Lactococcus fujiensis, Lactococcus galvier, Lactococcus lactis, such as Lactococcus lactis subspecies Cremoris, Lactococcus lactis subspecies Holdoniae, Includes, but is not limited to, Lactococcus lactis subspecies Lactoccus, or Lactococcus lactis subspecies Lactococcus, Lactococcus pissium, Lactococcus plantarum, Lactococcus raffinolactis, or Lactococcus tywanensis.

Preferably, the bacterium is a bacterium classified as "generally recognized safe" (GRAS) of the genus Lactococcus, Lactococcus lactis subspecies Cremoris, Lactococcus lactis subspecies Lactis, and Lactococcus. • Includes, but is not limited to, raffinolactis.

Preferably, the bacterium is Lactococcus lactis, preferably Lactococcus lactis subspecies Lactis, Lactococcus lactis subspecies Lactis biovariant diacetilactis, or Lactococcus lactis subspecies Cremoris, more preferably Lactococcus lactis subspecies. Seed Cremoris.

In another embodiment, the bacterium is the genus Leuconostoc sp. From Leuconostoc Ameliebiosam, Leuconostoc Argentinam, Leuconostoc Carnosam, Leuconostoc Citreum, Leuconostoc Cremoris, Leuconostoc dextranicum, Leuconostoc dulionis, Leuconostoc Stock Farax, Leuconostoc ficulneum, Leuconostoc fructosum, Leuconostoc gashikomitertam, Leuconostoc geridam, such as Leuconostoc geridam subspecies Enigmaticum, Leuconostoc geridam subspecies gashikomitertam, Or Leuconostoc Geridam subspecies Geridam, Leuconostoc Holtzapferii, Leuconostoc Inhae, Leuconostoc Kimchii, Leuconostoc Lactis, Leuconostoc mecenteroides, for example Leuconostoc mecenteroides sub Species Cremoris, Leuconostoc mecenteroides subspecies Dextranicamus, Leuconostoc mecenteroides subspecies Mecenteroides, or Leuconostoc mecenteroides subspecies Sionicum, Leuconostoc Miyukimuchii, Leuconostoc Oeni, Leuconostoc parame Includes, but is not limited to, Centeroides, Leuconostoc pseudoficurneum, or Leuconostoc pseudomesenteroides.

Preferably, the bacterium is the genus Leuconostoc sp. A bacterium classified as "generally recognized as safe" (GRAS) in Leuconostoc Carnosam, Leuconostoc citreum, Leuconostoc Farax, Leuconostoc Holtzapferii, Leuconostoc Inhae, Leuconostoc Kimchii, Leuconostoc Lactis, Leuconostoc Mecenteroides subspecies Cremoris, Leuconostoc Mecenteroides subspecies Dextranicamus, Leuconostoc Mecenteroides subspecies Mecenteroides, Leuconostoc • Includes, but is not limited to, Palmae, or Leuconostoc Pseudomesenteroides.

Preferably, the bacterium is the genus Leuconostoc sp. A bacterium with the "Safety Eligibility Estimate" (QPS) status of Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mecenteroides subspecies Cremoris, Leuconostoc mecenteroides subspecies Dextranicum, or Leuconostoc. Includes, but is not limited to, Stock Mecenteroides subspecies Mecenteroides.

In another embodiment, the bacterium is Pediococcus sp. From the genus Pediococcus acidilactisi, Pediococcus Argentinas, Pediococcus sericola, Pediococcus Clausenii, Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus etanoli Durance, Pediococcus Harophilus, Pediococcus Inopinatas, Pediococcus Lori, Pediococcus Pulbles, Pediococcus Pentosaseus, Pediococcus Siamensis, Pediococcus Stillesii, or Pediococcus Ulinas However, but not limited to these.

Preferably, the bacterium is the genus Pediococcus sp. Bacteria with a "Safety Eligibility Estimate" (QPS) status, including, but not limited to, Pediococcus acidilactisi, Pediococcus dextrinicus, or Pediococcus pentosaceus.

In another embodiment, the bacterium is the genus Propionibacterium sp. From Propionibacterium acidibacterium, Propionibacterium acidipropionichi, Propionibacterium acnes, Propionibacterium australians, Propionibacterium avidum, Propionibacterium Cyclohexanicam, Propionibacterium damnotham, Propionibacterium fludenreich, for example Propionibacterium fludenreich subspecies fludenreich or propionibacterium fludenle Ichii subspecies Shamanii, Propionibacterium granulosum, Propionibacterium innocium, Propionibacterium gensenii, Propionibacterium phosphophyllum, Propionibacterium microaerophyllum, Propionibacterium olivae , Propionibacterium propionicum, or propionibacterium soeniy, but not limited to these.

According to certain embodiments, the bacterium is not Propionibacterium acnes.

More preferably, the bacterium is the genus Propionibacterium sp. Propionibacterium acidipropionichi, Propionibacterium fludenreich subspecies fludenreich, which is a bacterium classified as "generally recognized as safe" (GRAS). Includes, but is not limited to, Propionibacterium fludenreich subspecies Shamanii, Propionibacterium gensenii, or Propionibacterium soeni.

More preferably, the bacterium is Propionibacterium fludenreich, more preferably Propionibacterium fludenreich subspecies fludenreich, or propionibacterium fludenreich. It is a subspecies Shamanii.

In another embodiment, the bacterium is the genus Streptococcus sp. From Streptococcus acidminimus, Streptococcus ajusens, Streptococcus agaractiae, Streptococcus alactolytics, Streptococcus anginosus, Streptococcus australis, Streptococcus bovis, Streptococcus cavalis, Streptococcus cavalis Castreus, Streptococcus secolm, Streptococcus constellatas, such as Streptococcus constellatas subspecies Consteratas, Streptococcus constellatas subspecies Faringes, or Streptococcus constellatas subspecies Bibogensis, Streptococcus cremoris, Streptococcus cremoris Streptococcus danieliae, Streptococcus defectivas, Streptococcus dentapuri, Streptococcus dentilsetti, Streptococcus dentassini, Streptococcus dentisani, Streptococcus debriestis, Streptococcus debriestei, Streptococcus didelphisto Streptococcus disgalactiae, such as Streptococcus disgalactiae subspecies Disgalactiae or Streptococcus disgalactiae subspecies Equisimiris, Streptococcus enterricus, Streptococcus equii, such as Streptococcus equic subspecies Equi, Streptococcus. Subspecies Luminatram, or Streptococcus Equi subspecies Zoo Epidemicas, Streptococcus equinus, Streptococcus faecalis, Streptococcus fascium, Streptococcus fels, Streptococcus subspecies Streptococcus galinaseus, Streptococcus galinaceus, Streptococcus galinaceus, Streptococcus galinaceus Galoricus subspecies Macedonicas, or Streptococcus galoriticus subspecies Pasturianus, Streptococcus garvie, Streptococcus gordonii, Streptococcus ha Streptococcus hansenii, Streptococcus henry, Streptococcus honkongensis, Streptococcus hyointestinalis, Streptococcus hyobaginalis, Streptococcus ictalli, Streptococcus sub-Streptococcus infantalis, for example Streptococcus infantalis Fantarius subspecies Infantarius, Streptococcus infantis, Streptococcus iniae, Streptococcus intermedius, Streptococcus intestinaris, Streptococcus lactrius, Streptococcus lactis, Streptococcus lactis subspecies, for example Streptococcus lactis subspecies Diacetylactis, or Streptococcus lactis subspecies Lactis, Streptococcus loxodontisalivarius, Streptococcus lutetiensis, Streptococcus macacae, Streptococcus macedonicas, Streptococcus macedonicas, Streptococcus marimanmarium, Streptococcus , Streptococcus morbilolm, Streptococcus morosensis, Streptococcus mutans, Streptococcus oligofermentans, Streptococcus olaris, Streptococcus orisasini, Streptococcus oristoccus, Streptococcus oristococcus・ Pasturianus, Streptococcus perolis, Streptococcus phocae, such as Streptococcus phocae subspecies Fokae or Streptococcus phocae subspecies Salmonis, Streptococcus plantarum, Streptococcus preomorphus, Streptococcus preomorphus, Streptococcus plulococcus Porsi, Streptococcus porcinus, Streptococcus porcolum, Streptococcus pseudoneumonier, Streptococcus pseudoporcinus, Streptococcus pi Ogenes, Streptococcus rafinolactis, Streptococcus ratti, Streptococcus rifensis, Streptococcus rubnelli, Streptococcus lupiculae, Streptococcus saccharoriticus, Streptococcus subvariant, Streptococcus salivarius, for example Streptococcus salivarius.・ Salibirozodontae, Streptococcus sanginis, Streptococcus shiroi, Streptococcus sinensis, Streptococcus sobrinus, Streptococcus swiss, Streptococcus thermophilus, Streptococcus soltensis, Streptococcus tyrcuris , Streptococcus ursolis, Streptococcus bestibularis, or Streptococcus Wyus, but not limited to these.

Preferably, the bacterium is the genus Streptococcus sp. Classified as "generally recognized as safe" (GRAS), including Streptococcus thermophilus strain Th4, Streptococcus galoriticus subspecies Macedonicas, Streptococcus salivarius subspecies Sullivarius, or Streptococcus salivarius subspecies Thermophilus. Not limited to these.

Preferably, the bacterium is the genus Streptococcus sp. Bacteria with a "Safety Eligibility Estimate" (QPS) status, including, but not limited to, Streptococcus thermophilus.

In a preferred embodiment, the bacterium does not produce endotoxin or other potentially toxic substance. Thereby, the bacteria are safe to use and are not harmful to the subject after application.

Preferably, the bacterium does not produce spores. Bacterial spores are not part of the reproductive cycle, but are resistant structures used for survival under adverse conditions. Recombinant bacteria preferably do not produce spores, so that recombinant bacteria cannot survive, for example, without nutrients or in the absence of auxotrophic factors.

Preferably, the bacteria do not produce inclusion bodies. Inclusion bodies often contain overexpressed proteins, and aggregation of overexpressed proteins in inclusion bodies can be irreversible.

Bacteria used to obtain recombinant bacteria of the invention preferably do not produce inclusion bodies, so the amount of at least one heterologous factor after transcription and preferably translation of each nucleic acid sequence is intracellular in the inclusion bodies. It does not gradually decrease due to accumulation.

More preferably, the bacterium also does not produce extracellular proteinase. Extracellular proteinases are secreted by bacteria and can disrupt extracellular structures such as proteins to produce nutrients such as carbon, nitrogen, or sulfur. Extracellular proteinases can also act as exotoxins and may be examples of virulence factors for bacterial pathogenesis.

Due to the absence of extracellular proteinase, the safety of the bacterium after application to the subject is preferably increased. Moreover, the bacterium preferably does not degrade at least one heterologous factor after release from the recombinant bacterium.

In a more preferred embodiment, the recombinant bacterium is a lactic acid bacterium, preferably a Lactobacillus or Lactococcus species. In a more preferred embodiment, the Lactococcus species is the Lactococcus lactis subspecies Cremoris.

Lactic acid bacteria also release lactic acid as a major metabolic end product of carbohydrate fermentation. Lactic acid is also known to stimulate endothelial growth and proliferation. Moreover, lactic acid has an antibacterial effect, which reduces the probability of bacterial infection at the site of skin damage.

Techniques for transforming the bacteria mentioned above are known to those of skill in the art, such as Green and Sambrook (2012): "Molecular coloring: a laboratory manual", fourth edition, Cold Spring Harbor Laboratory. Harbor, NY, US).

After transformation, the recombinant bacterium is functionally coupled to a prokaryotic chloride-inducible promoter and has at least one nucleic acid sequence encoding at least one heterologous factor, the heterologous factor independently heterologous poly. A peptide or complex thereof, comprising at least one prokaryotic regulator gene that controls the activity of a chloride-inducible promoter, wherein the heterologous polypeptide is at least one eukaryotic polypeptide, at least one thereof. Includes two fragments, or a combination thereof.

More preferably, the recombinant bacterium contains at least one copy of the recombinant plasmid of the invention after transformation.

Suitable strains for obtaining the recombinant bacterium of the present invention are commercially available, for example, from MoBiTec GmbH (Göttingen, Germany) or from NIZO food research BV (Ede, NL). Suitable strains are, for example, Lactococcus lactis strain NZ1330, which preferably comprises a plasmid selection system that is not based on antibiotic resistance.

In a more preferred embodiment, the nucleic acid sequence encoding each heterologous factor is modified to increase the expression, secretion, and / or stability of at least one heterologous factor encoded.

Suitable modifications are known to those of skill in the art and include, for example, adapting the codon frequency of at least one nucleic acid sequence to the bacterium used.

For example, at least one nucleic acid sequence may be designed to fit the codon usage pattern of the bacterium used. Furthermore, in addition to general codon optimization, a specific codon table, such as the codon table of the highly expressed ribosomal protein gene of each bacterium, is used to translate at least one heterologous factor encoded. It can be further increased.

Suitable modifications also include, for example, integration of the nucleic acid sequence encoding the secretory signal sequence at the 5'end of the open reading frame (ORF) of the heterologous factor, creating a fusion protein containing the secretory signal sequence and the heterologous factor. .. The secretory signal sequence is preferably located on the N-terminal side of each heterologous factor.

The secretory signal sequence preferably directs the newly synthesized protein to the plasma membrane. The secretory signal sequence, preferably at the end of the signal peptide, is preferably recognized and cleaved by a signal peptidase to produce a free secretory signal sequence, preferably the signal peptide, and a mature heterologous factor secreted extracellularly. There is a stretch of amino acids.

Suitable secretory signal sequences include, for example, signal peptides.

Preferably, the secretory signal sequence is preferably the native signal peptide of each factor encoded by the nucleic acid sequence that is the precursor of each heterologous factor.

In a more preferred embodiment, the secretory signal sequence is a homologous secretory sequence from a recombinant bacterium, preferably the secretory signal sequence is Lactococcus sp. Unknown secretory protein 45 (Usp45) signal sequence. The Usp45 secretory signal has a very high secretory efficiency. Other secretory signals are known to those of skill in the art, such as Lactococcus sp. Includes PrtP, SlpA, SP310, SPEXP4, and AL9.

The amino acid sequence of the Usp45 protein of the Lactococcus lactis subspecies Cremoris MG1363 is available, for example, at GenBank Accession No. CAL99070.1. The signal sequence spans amino acids 1-27 of the sequence deposited at GenBank Accession No. CAL99070.1.

For example, the native signal peptide of a heterologous factor can be replaced by a homologous secretory signal sequence from a recombinant bacterium used to express at least one heterologous factor.

The secretory signal sequence preferably improves the secretory efficiency of each heterologous factor. For example, in Lactococcus lactis, most proteins are secreted by the Sec pathway. The protein is synthesized as a precursor containing the mature portion of the protein with an N-terminal signal peptide. The signal peptide targets the protein to the cytoplasmic membrane and aids in the secretion of the protein. After cleavage of the signal peptide, the mature protein is released extracellularly.

More preferably, the secretory signal comprises the amino acid sequence of SEQ ID NO: 3, which is also illustrated in FIG.

In a more preferred embodiment, at least one heterologous factor, preferably 1, 2, 3, 4, or more heterologous factors (s) are expressed as propeptides with or without secretagogues. The propeptide can be cleaved by a protease to release a mature form of at least one heterologous factor, preferably one, two, three, four, or more heterologous factors (s).

Preferably, at least one heterologous factor, preferably one, two, three, four, or more heterologous factors (s) can be released from the recombinant bacterium.

For example, in a gram-positive bacterium such as a lactic acid bacterium, the protein to be secreted from the bacterium is preferably synthesized as a precursor containing an N-terminal signal peptide and a mature portion of the protein. Precursors are recognized by the bacterial secretory mechanism and translocate through the cytoplasmic membrane. The signal peptide is then cleaved and degraded, and the mature protein is released from the bacterium, for example to the culture supernatant, or to the area of inflammatory skin damage.

For example, Lactococcus lactis can secrete proteins by a Sec-dependent pathway, ranging from low molecular weights, eg, less than 10 kDa, to high molecular weights, eg, molecular weights above 160 kDa.

Alternatively, at least one heterologous factor, preferably one, two, three, four, or more heterologous factors (s) are released from the recombinant bacterium through the leaky cytoplasmic membrane.

For example, the cytoplasmic membrane of recombinant bacteria is vulnerable to leakage due to damaged synthesis of cell wall components.

Preferably, the recombinant bacterium comprises an inactivated alanine racemase (all) gene. In the absence of D-alanine, recombinant bacteria cannot maintain the integrity of the cytoplasmic membrane, and after that, at least one heterologous factor, preferably 1, 2, 3, 4, or more heterologous factors (singular or). Multiple) are released from recombinant bacteria.

In a more preferred embodiment, the recombinant bacterium of the invention comprises at least one inactivated gene encoding an essential protein required for bacterial viability.

In a preferred embodiment, the essential protein required for the viability of the recombinant bacterium is a protein required for the synthesis of the organic compound required for the viability of the recombinant bacterium, more preferably an enzyme.

After inactivation of essential proteins, preferably enzymes, each organic compound is required for the viability of the recombinant bacterium and must be supplemented with auxotrophic factors to sustain the viability of the recombinant bacterium. Is.

Preferably, the auxotrophic factor is a vitamin, amino acid, nucleic acid, and / or fatty acid.

For example, amino acids and nucleotides are biologically important organic compounds, which are precursors of proteins and nucleic acids, respectively.

Preferably, the gene encoding the essential protein required for bacterial viability is provided on recombinant nucleic acids and / or recombinant plasmids according to the invention.

In a more preferred embodiment, the at least one gene required for the viability of the recombinant bacterium is alanine racemase (all), thymidylate synthase (thyA), aspartate synthase (asnH), CTP synthase (pyrG), tryptophan synthase ( It is selected from the group consisting of trbBA) and combinations thereof.

For example, alanine racemase is an enzyme that catalyzes the racemization of L-alanine to L- and D-alanine. Alanine D-alanine produced by lasemase is used for peptidoglycan synthesis. Peptidoglycan is found in the cell walls of all bacteria.

It is known to those of skill in the art that inactivating the bacterial alanine racemase (all) gene results in the need for the bacterium to use an external source of D-alanine to maintain cell wall integrity.

For example, Lactococcus lactis and Lactobacillus plantarum are known to those of skill in the art to contain a single aller gene. Gene inactivation affects the integration of D-alanine into the cell wall.

For example, Lactococcus lactis bacteria carrying the inactivated alanine racemase (all) gene can synthesize peptidoglycan and integrate D-alanine into lipoteichoic acid (LTA) in order for D-alanine. Depends on external supply. These bacteria dissolve rapidly when D-alanine is removed, for example in mid-exponential growth.

Alanine racemase-deficient strains of lactic acid bacteria can be produced, for example, by methods known in the art.

Thymidine monophosphate is an enzyme that catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidylate monophosphate (dTPP). dTPM is one of the three nucleotides that form thymine (dTPP, dTDP, and dTTP). Thymidine is a nucleic acid of DNA.

It is known to those of skill in the art that thymidylate synthase plays a significant role in DNA biosynthesis. Moreover, bacterial growth requires the synthesis of DNA.

Inactivation of the thymidylate synthase (thyA) gene in the bacterium results in the need for the bacterium to use an external source of thymidine to maintain DNA integrity.

Asparagine synthetase is an enzyme that produces the amino acid asparagine from aspartic acid. Inactivation of the asparagine synthetase (asnH) gene results in the inability to synthesize each amino acid, which becomes an auxotrophic factor.

CTP synthase is an enzyme involved in pyrimidine synthesis. CTP synthase interconverts uridine-5'-triphosphate (UTP) and cytidine-5'-triphosphate (CTP). Inactivation of the CTP synthase (pyrG) gene prevents bacteria from synthesizing cytosine nucleotides from both de novo synthesis and the uridine cell wall pathway.

CTP becomes an auxotrophic factor, which must be supplemented for RNA and DNA synthesis.

Tryptophan synthase is an enzyme that catalyzes the last two steps of the biosynthesis of the amino acid tryptophan.

Inactivation of the tryptophan synthase (trpBA) gene prevents bacteria from synthesizing their respective amino acids, which becomes an auxotrophic factor.

Methods for inactivating genes required for bacterial viability are known to those of skill in the art and are mediated by gene deletions, gene mutations, and gene RNA interference (RNAi) -mediated gene silencing. , Inhibition of gene translation, or combinations thereof.

In a more preferred embodiment, each auxotrophic factor is provided with the recombinant bacterium.

In a more preferred embodiment of the invention, the inactivated gene required for the viability of the recombinant bacterium is used for environmental containment.

After application of the recombinant bacterium containing at least one inactivated gene required for bacterial viability, each auxotrophic factor may be externally administered, eg, reprepared medium, application medium, or growth medium. Must be provided.

In cases where the recombinant bacterium is released into the environment, the auxotrophic factor is lacking, preferably the recombinant bacterium dies.

Moreover, the application of externally supplemented auxotrophic factors allows control of the biosynthesis and release of each heterologous factor (s).

More preferably, a bacterium selected from the group consisting of alanine racemase (all), thymidylate synthase (thyA), aspartate synthase (asnH), CTP synthase (pyrG), tryptophan synthase (trbBA), and combinations thereof. Genes encoding essential proteins required for viability are provided on recombinant nucleic acids and / or recombinant plasmids according to the invention.

Thereby, it is possible to provide a highly stable system for maintaining recombinant nucleic acids and / or recombinant plasmids according to the invention in recombinant bacteria, even during in vivo growth, because it is preferably external. This is because the selective pressure for maintaining the recombinant nucleic acid and / or the recombinant plasmid according to the present invention can be achieved in the absence of the nutritional requirement factor supplemented with.

Preferred for viability of bacteria selected from the group consisting of alanin plasmid (all), thymidylate synthase (thyA), asparagine synthase (asnH), CTP synthase (pyrG), tryptophan synthase (trbBA), and combinations thereof. Expression of the gene encoding the essential protein required for the present invention not only allows complementation of nutritional requirements on the recombinant nucleic acid and / or recombinant plasmid according to the present invention, but also allows the recombinant nucleic acid and / or recombinant according to the present invention in each bacterium. It can also be used as a selection marker for the presence of a plasmid.

According to a preferred embodiment, the recombinant bacterium should be used medically, more preferably for use in the treatment of chronic inflammatory wounds or degenerative conditions, or for the treatment of tumors, preferably malignant tumors. For use.

During administration of the recombinant bacterium of the invention to a subject, at least one heterologous factor, preferably one, two, three, four, or more heterologous factors (s) are released, eg, by cleavage of proteinase. It can undergo degradation, which can lead to loss of biological activity of the heterologous factor (s).

Degradation or depletion of the heterologous factor (s) is at least one heterologous factor (preferably 1, 2, 3, 4, or more) from the recombinant bacterium of the invention in the presence of chloride ions. Can be compensated for by continuous release (s).

Preferably, the recombinant bacterium expresses at least one heterologous factor, preferably 1, 2, 3, 4, or more heterologous factors (s) in a certain manner. Preferably, the recombinant bacterium releases a constant release of heterologous factors (s).

The pharmaceutical composition of the invention comprises a recombinant bacterium according to the invention and at least one pharmaceutically acceptable excipient.

In a preferred embodiment, the recombinant bacterium of the invention is in a pharmaceutical composition for medical use, more preferably for the treatment of chronic inflammatory wounds or degenerative conditions, or for tumors. , Preferably provided for use in the treatment of malignant tumors.

Excipients are substances that are formulated with the active ingredient of the drug and are long-term stabilizing, increasing the amount to a solid formulation containing a small amount of the potent active ingredient (hence, in many cases, " To impart a therapeutic enhancement to the active ingredient in the bulking agent, "filler", or "diluent"), or the final dosage form, eg, to aid drug absorption, to reduce viscosity, Alternatively, it is included for the purpose of enhancing solubility.

The term "excipient" preferably also includes at least one diluent.

For example, to support in vitro stability, eg, to support the handling of the active substance by helping powder fluidity or non-adhesive properties, in addition to preventing denaturation or aggregation during the expected quality retention period. Excipients may also be useful in the manufacturing process, and / or to contribute to the viability of the bacterial cells of the recombinant bacteria during freezing and thawing.

Preferably, the pharmaceutical composition further preferably comprises at least one pharmaceutically acceptable excipient suitable for the intended route of administration.

Preferably, the pharmaceutically acceptable excipient is known to those of skill in the art and comprises at least one polymer carrier, preferably selected from the group consisting of polysaccharides, polyesters, polymethacrylamides, and mixtures thereof. For example, a pharmaceutically acceptable excipient is hydrogel, which, for example, provides an optimal moist environment for promoting wound healing, for example. Furthermore, pharmaceutically acceptable excipients, after application, preferably aid the adhesion of recombinant bacteria to the site of a chronic inflammatory wound or degenerative condition, or tumor, preferably malignant tumor.

Preferably, the pharmaceutical composition further comprises at least one nutrient for the recombinant bacterium and is preferably selected from the group consisting of carbohydrates, vitamins, minerals, amino acids, trace elements, and mixtures thereof.

More preferably, the pharmaceutical composition further stabilizes at least one, preferably 1, 2, 3, 4, or more heterologous factors (s) and / or the recombinant bacterium of the invention. Includes at least one component. Stabilizing compounds are preferably metal cations, preferably divalent metal cations such as Mg ²⁺ , Ca ²⁺ , and mixtures thereof, antimicrobial agents, cryoprotectants, protease inhibitors, reducing agents, metal chelating agents, and It is selected from the group consisting of a mixture thereof.

In a preferred embodiment, the recombinant bacterium of the invention is in the form of a kit according to the invention for medical use, more preferably for the treatment of chronic inflammatory wounds or degenerative conditions. , Or for the treatment of tumors, preferably malignant tumors, the recombinant bacterium can express at least one heterologous factor under the control of a chloride-inducible promoter of the prokaryotic system, and the kit further comprises. Includes at least one inducing factor, including chloride ions.

Preferably, the kit is used as a combination preparation for separate, sequential or simultaneous use in medicine, more preferably for the treatment of chronic inflammatory wounds or degenerative conditions. Or for the treatment of tumors, preferably malignant tumors.

Preferably, the inducing factor containing chloride ions is in the form of a liquid containing chloride ions, preferably in the form of a culture medium, more preferably with at least one of the previously mentioned pharmaceutically acceptable excipients. Provided to.

In a preferred embodiment, the recombinant bacterium of the invention is in the form of a medical device of the invention for medical use, more preferably for the treatment of chronic inflammatory wounds or degenerative conditions. Provided for the treatment of tumors, preferably malignant tumors, or recombinant bacteria can express at least one heterologous factor under the control of a prokaryotic chloride-inducible promoter.

Suitable medical devices preferably further include at least one support and / or container for the provision of recombinant bacteria, such as cell encapsulation systems and / or microbeads.

Suitable medical devices are preferably stents, implants, inhalers, encapsulation systems, or medical dressings.

Preferably, the pharmaceutical composition and / or kit and / or medical device according to the invention comprises a solution, lyophilized or dried, preferably lyophilized or spray dried recombinant bacterium. More preferably, the recombinant bacterium is reprepared with a solution containing chloride ions, preferably a culture medium, prior to administration of the recombinant bacterium and / or the pharmaceutical composition and / or kit to the subject.

The recombinant bacterium of the invention is preferably contacted with at least one inducing factor to induce expression of at least one, preferably one, two, three, four, or more heterologous factors (s). Can be made to.

Further, preferably, after induction of expression, at least one, preferably 1, 2, 3, 4, or more heterologous factors (s) are released from the bacterium.

Preferably, at least one, preferably 1, 2, 3, 4, or more heterologous factors (s) are expressed with a secretory signal sequence, preferably an N-terminal signal peptide. After expression of each factor, the secretory signal sequence, preferably the N-terminal signal peptide, can be removed. Alternatively, the first, second, and / or third heterologous factors can be expressed in mature form, preferably without a secretory signal sequence, preferably an N-terminal signal peptide.

The repreparation medium containing chloride ions used to reprepare recombinant bacteria according to the invention can be in the form of a solution or dispersion, such as an emulsion, suspension, gel, preferably hydrogel, or colloidal solution. ..

Preferably, the reprepared medium contains chloride ions in an amount sufficient to initiate expression of at least one heterologous factor under the control of a prokaryotic chloride-inducible promoter.

Suitable polymers for obtaining gels, preferably hydrogels, are known to those of skill in the art and include natural and / or synthetic polymers.

Preferably, the recombinant bacterium of the invention is provided in an effective amount to treat and / or alleviate the process of the disease of interest, such as preferably a chronic inflammatory wound, or degenerative condition, or tumor, preferably a malignant tumor. To.

Preferably, the pharmaceutical composition and / or kit and / or medical device according to the invention comprises an effective amount of the recombinant bacterium of the invention.

In a preferred embodiment of the invention, the recombinant bacterium of the invention and / or the pharmaceutical composition and / or kit and / or medical device according to the invention should be used for the treatment of inflammatory wounds, preferably chronic inflammatory wounds. be.

In a preferred embodiment, the inflammatory, preferably chronic inflammatory wound preferably comprises frostbite, dermatitis, ulcers, and combinations thereof, more preferably ulcers.

Inflammatory wounds can also include inflammatory skin trauma, which can progress to a chronic inflammatory condition.

Frostbite is a medical condition in which localized damage is caused to the skin and other tissues due to freezing and may involve tissue destruction. Frostbite can also be chilblains, which are superficial ulcers of the skin resulting from exposure to cold and moisture. Damage to the capillary bed of the skin causes redness, itching, inflammation, and in some cases blisters. More preferably, the frostbite is chilblains.

Chronic inflammatory wounds are known to those of skill in the art and include, for example, chronic venous ulcers, chronic arterial ulcers, chronic diabetic ulcers, and chronic pressure ulcers. Preferably, the chronic wound is at least one of chronic venous ulcer, chronic arterial ulcer, chronic decubitus, and their chronic pre-ulcerization stage, preferably chronic venous ulcer, chronic arterial ulcer, and chronic decubitus. be.

Chronic wounds occur in various morphologies and prevalences. Chronic wounds do not heal in an orderly set of stages and in a predictable amount of time compared to acute wounds. Wounds that do not heal within 1-3 months or do not respond to the initial treatment are typically classified as chronic. In acute wounds, there is a good balance between molecular production and degradation in the microenvironment as well as immune cell activation levels, leading to a well-controlled, step-by-step healing process. In chronic wounds, this balance is lost, resulting in the breakdown and abnormal activation of the local immune system. Chronic wounds usually remain in one or more phases of the wound healing process, eg, the inflammatory stage. This type of wound is referred to as a chronic inflammatory wound.

Chronic wounds are usually the result of various underlying illnesses and medical conditions such as tumors, diabetes (causing DFU), poor blood circulation (causing VLU), surgery, and burns. Ischemia (ie, lack of oxygen in the extremities), bacterial infection and colonization, increased proteolytic enzymes, and inflammation are the usual underlying pathophysiological causes of refractory wounds.

The ulcer can be a decubitus ulcer or a leg ulcer. Ulcers can also be venous ulcers, arterial ulcers, diabetic ulcers, or pressure ulcers. Ulcers can also be the pre-ulcerization stage of the ulcers mentioned above, without any visible sign of open skin wounds. Without medical intervention, the pre-ulcerization stage can progress to ulceration.

Chronic venous ulcers can usually occur in the legs, affecting about 70% to 90% of chronic wounds and usually affecting older patients.

Another major cause of chronic inflammatory wounds is diabetes. Patients with diabetes have a 15% higher risk of amputation than the general population due to chronic ulcers. Diabetes causes neuropathy, which impairs nociception and pain perception. Therefore, the patient may be unaware of small wounds on the legs and feet, and thus may fail to prevent infection or recurrent injury.

A further problem is that diabetes causes immunosuppression and damage to small blood vessels, resulting in reduced oxygenation of tissues. Preventing sufficient tissue oxygenation significantly increases the prevalence of chronic inflammatory wounds.

Pressure sores, also known as decubitus ulcers or bedsores, can occur with or without a diabetic condition. Pressure ulcers are injuries localized to the skin and / or underlying tissue, which can occur in bone protrusions as a result of compression, or compression in combination with shear or rubbing.

Currently, standard treatment management for chronic inflammatory wounds, including diabetic wounds such as leg ulceration, is primarily focused on controlling infection and promoting revascularization. Despite these strategies, the rate of amputation remains unacceptably high in patients with leg ulceration.

Moreover, it is present when the underlying disease state or cause of ulceration, such as diabetes or chronic venous insufficiency, is ameliorated and / or treated, for example by controlling blood glucose levels or by administering blood pressure medications, respectively. Ulcers can still take a very long time to heal.

Therefore, to reactivate and promote wound healing in patients with chronic wounds to overcome the lack of definitive non-invasive treatment of chronic inflammatory wounds, including diabetic wounds such as leg ulceration. New strategies are urgently needed.

Preferably, in the case of inflammatory wounds, preferably chronic inflammatory wounds, a unique combination of factors, preferably released from bacteria, allows reprogramming of chronic inflammatory wounds into acute wounds, which is the wound afterwards. Receive closure.

In a preferred embodiment, in the case of inflammatory wounds, preferably chronic inflammatory wounds, the recombinant bacterium should be administered systemically, topically and / or even more preferably topically by subcutaneous injection. ..

Recombinant bacteria should preferably be administered topically to inflammatory, preferably chronic inflammatory wounds to be treated.

Recombinant bacteria are applied to inflammatory, preferably chronic inflammatory wounds, locally and / or to the vicinity of the wound, preferably to the edges or cavities of inflammatory, preferably chronic inflammatory wounds. Can be administered by subcutaneous injection of.

Moreover, topical application or subcutaneous injection of the recombinant bacterium of the invention to and / or within the site of pre-ulceration can prevent progression from pre-ulceration to open inflammatory wounds.

In a preferred embodiment, in the case of a tumor, preferably a malignant tumor, the recombinant bacterium is administered systemically, eg, by intravenous injection, or locally, preferably by intratumoral injection, and / or by intraperitoneal injection. Should be.

The recombinant bacterium is preferably administered to a tumor, preferably a malignant tumor, by intratumoral injection into the tumor of the recombinant bacterium of the present invention and / or by injection into the vicinity of the tumor and / or by intraperitoneal injection. ..

In a preferred embodiment, the recombinant bacterium according to claim 1 encodes at least one nucleic acid sequence encoding a first heterologous factor, at least one nucleic acid sequence encoding a second heterologous factor, and a third heterologous factor. It contains at least one nucleic acid sequence, provided that the first factor, the second factor, and the third factor are functionally different from each other, the first factor being a growth factor and the second factor being. It is selected from the group consisting of M2 polarization factors, and the third factor is selected from the group consisting of M2 polarization factors and growth factors.

In a preferred embodiment, the second and third heterologous factors are selected from the group consisting of M2 polarization factors, the second factor and the third factor being functionally different M2 polarization factors.

That is, the second heterologous factor is the first M2 polarization factor, and the third heterogeneous factor is the second M2 polarization factor that is functionally different from the first M2 polarization factor.

Preferably, the first M2 polarization factor is colony stimulating factor-1 (CSF-1), interleukin 34 (IL-34), interleukin 4 (IL-4), and interleukin 13 (IL-13). It is an M2 polarization factor selected from the group consisting of, and the second M2 polarization factor is colony stimulating factor-1 (CSF-1), interleukin 34 (IL-34), interleukin 4 (IL-4). ), Interleukin 10 (IL-10), and interleukin 13 (IL-13). It is functionally different from the chemical factor.

More preferably, the first M2 polarization factor is a colony stimulating factor-1 receptor (CSF1R) ligand and the second M2 polarization factor is interleukin 4 (IL-4), interleukin 10 (IL-). 10), an M2 polarization factor selected from the group consisting of interleukin 13 (IL-13), their functional analogs, their biosimilars, and mixtures thereof.

A more preferred combination of M2 polarization factors is:
-Colony-stimulating factor-1 and interleukin-4,
-Colony-stimulating factor-1 and interleukin-13,
-Colony-stimulating factor-1 and interleukin-10,
Interleukin-34 and Interleukin-4,
Interleukin-34 and interleukin-13,
Interleukin-34 and Interleukin-10,
Interleukin-4 and Interleukin-10, or Interleukin-13 and Interleukin-10,
Is.

A more preferred combination mentioned above for M2 polarization factors is at least one of the growth factors mentioned above, preferably fibroblast growth factor 1, fibroblast growth factor 2, fibroblast growth. Factor 7, fibroblast growth factor 10, hepatocellular growth factor, transforming growth factor beta (TGF-beta), epithelial growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor- Combined with a growth factor selected from the group consisting of α (TGF-α) and platelet-derived growth factor BB.

Preferably, the first, second, and third heterologous factors are
Fibroblast growth factor 2, colony stimulating factor-1, and interleukin-4,
Fibroblast Growth Factor 2, Interleukin-34, and Interleukin-4,
Fibroblast growth factor 2, colony stimulating factor-1, and interleukin-13,
Fibroblast Growth Factor 2, Interleukin-34, and Interleukin-13,
Fibroblast growth factor 2, colony stimulating factor-1, and interleukin-10,
Fibroblast Growth Factor 2, Interleukin-34, and Interleukin-10,
Fibroblast growth factor 7, colony stimulating factor-1, and interleukin-4,
Fibroblast Growth Factor 7, Interleukin-34, and Interleukin-4,
Fibroblast growth factor 7, colony stimulating factor-1, and interleukin-13,
Fibroblast Growth Factor 7, Interleukin-34, and Interleukin-13,
Fibroblast growth factor 7, colony stimulating factor-1, and interleukin-10,
Fibroblast Growth Factor 7, Interleukin-34, and Interleukin-10,
Transforming Growth Factor Beta, Colony Stimulating Factor-1, and Interleukin-4,
Transforming Growth Factor Beta, Interleukin-34, and Interleukin-4,
Transforming Growth Factor Beta, Colony Stimulating Factor-1, and Interleukin-13,
Transforming Growth Factor Beta, Interleukin-34, and Interleukin-13,
Transforming Growth Factor Beta, Colony Stimulating Factor-1, and Interleukin-10,
Transforming Growth Factor Beta, Interleukin-34, and Interleukin-10,
Transforming Growth Factor Alpha, Colony Stimulating Factor-1, and Interleukin-4,
Transforming Growth Factor Alpha, Interleukin-34, and Interleukin-4,
Transforming Growth Factor Alpha, Colony Stimulating Factor-1, and Interleukin-13,
Transforming Growth Factor Alpha, Interleukin-34, and Interleukin-13,
Transforming Growth Factor Alpha, Colony Stimulating Factor-1, and Interleukin-10,
Transforming Growth Factor Alpha, Interleukin-34, and Interleukin 10,
Platelet-derived growth factor BB, colony-stimulating factor-1, and interleukin-4,
Platelet-derived growth factor BB, interleukin-34, and interleukin-4,
Platelet-derived growth factor BB, colony-stimulating factor-1, and interleukin-13,
Platelet-derived growth factor BB, interleukin-34, and interleukin-13,
Platelet-derived growth factor BB, colony-stimulating factor-1, and interleukin-10, or platelet-derived growth factor BB, interleukin-34, and interleukin-10,
It is a combination of.

More preferably, the first, second, and third heterologous factors are fibroblast growth factor 2, colony stimulating factor-1, and interleukin-4, their functional analogs, and their biosimilars. It is a combination.

Preferably, release of two or more M2 polarization factors from the bacteria of the invention further results in M2 polarization of undifferentiated macrophages, M1 polarized macrophages, and undifferentiated monocytes, as well as other macrophage progenitor cells. Prompted.

In an alternative embodiment, the first factor is the first growth factor selected from the group consisting of the growth factors mentioned above and the third factor is the growth factor mentioned above. It is a second growth factor selected from the group consisting of, and functionally different from the first growth factor. Preferably, the second growth factor is transforming growth factor beta (TGF-beta).

More preferred combinations of growth factors are:
Fibroblast Growth Factor 1 and Transforming Growth Factor Beta,
Fibroblast Growth Factor 2 and Transforming Growth Factor Beta,
Fibroblast Growth Factor 7 and Transforming Growth Factor Beta,
Fibroblast Growth Factor 10 and Transforming Growth Factor Beta,
Platelet-derived growth factor BB and transforming growth factor beta,
Transforming Growth Factor Alpha and Transforming Growth Factor Beta,
Epidermal Growth Factor and Transforming Growth Factor Beta,
Heparin-binding EGF-like growth factor and transforming growth factor beta,
Hepatocyte growth factor and transforming growth factor beta, or vascular endothelial growth factor A and transforming growth factor beta,
Is.

The more preferred combinations mentioned above for growth factors are preferably colony stimulating factor-1 (CSF-1), interleukin-34 (IL-34), interleukin-4 (IL-4), interleukin. -10 (IL-10), interleukin-13 (IL-13), and mixtures thereof, preferably colony stimulating factor-1 (CSF-1), interleukin-34 (IL-34), interleukin-4. Combined with M2 polarization factor selected from the group consisting of (IL-4), interleukin-13 (IL-13), and mixtures thereof.

In another preferred embodiment of the invention, the recombinant bacterium of the invention and / or the pharmaceutical composition and / or kit and / or medical device according to the invention is a tumor, preferably a malignant tumor, preferably a peritoneal cancer, more preferably. Cancerous peritonitis, more preferably metastatic ovarian cancer, colorectal cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, bile sac cancer, renal cell carcinoma, transitional epithelial cancer, endometrial cervical cancer, and / or extraabdominal condition, It should be used, for example, in the treatment of cancer, lung cancer, and malignant melanoma.

Tumors are abnormal cell / tissue growth that has no physiology and is usually due to uncontrolled rapid cell proliferation. Tumors can be benign or malignant. Tumors of different types are named after the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas.

Peritoneal cancer can be divided into two categories; primary peritoneal cancer in which the primary tumor originates within the peritoneum, and cancerous peritonitis (PC) defined as intraperitoneal dissemination of any tumor originating elsewhere. ..

Cancerous peritonitis is one of the most common diffuse peritonitis, with ovarian cancer being the most common cause of it (46%), followed by colorectal cancer (31%), pancreatic cancer, gastric cancer, and liver. Cellular cancer, bile sac cancer, renal cell carcinoma, transitional epithelial cancer, endometrial, cervical cancer, and other malignancies including unknown primary. Extraabdominal conditions, such as breast cancer, lung cancer, and malignant melanoma, may involve the abdominal cavity via hematogenous spraying (Singh, S. et al. Research (2016), Volume 4, Issue 7, pages 735 to 748; doi: 10.21474 / IJAR01 / 936).

Tumor recurrence is confined to the abdominal cavity in 10% to 35% of patients with recurrent colorectal cancer (CRC) and up to 50% of patients with recurrent gastric cancer (GC) (Coccolini, F. et al., World J. Gastroenterol. 2013; 19 (41): patients 6979 to 6994, doi: 10.3748 / wjg.v19.I41.6979).

Peritoneal metastases can also be formed due to extraperitoneal cancers such as pleural mesothelioma, milk, and distant lung metastases. Colonization of tumor cells in the diaphragm or abdominal lymphatic vessels causes obstruction of lymphatic drainage and reduced outflow of peritoneal fluid, leading to cancerous disease and / or formation of ascites.

The immune system preferably plays a major role in the pathophysiology of cancer. Many tumors are severely infiltrated by different types of immune cells. However, the composition of these cells within the tumor can vary. Both cells from the adaptive immune system, such as cytotoxic and regulatory T cells, and cells from the innate immune system, such as macrophages, dendritic cells, and innate killer (NK) cells, are central to disease progression. It is a target existence. These immune cells appear to be linked to tumor growth, infiltration, and metastasis.

In particular, tumor growth parallels the recruitment and accumulation of macrophages, and these cells acquire the function of supporting tumor growth from primary tumors and dissemination of cancer cells. Moreover, there is clinical evidence demonstrating a tight correlation between increased numbers of macrophages with disease progression and poor prognosis. Since macrophages can produce different cytokines (Th1 and Th2-related cytokines) with a wide spectrum, they are central to the orchestration of the immune microenvironment.

Macrophages can be divided into M1 (classically activated) or M2 (selectively activated) phenotypes. M1 macrophages exhibit an pro-inflammatory phenotype and are characterized by the expression of pro-inflammatory cytokines such as IL-1, IL-6, TNF-α, and IFN-γ. Monocytes can be differentiated into M1-type macrophages by bacterial components such as lipopolysaccharide (LPS) and interferon gamma (INF-γ).

In contrast, M2 macrophages have an immunosuppressive phenotype and release factors that promote the Th2 response, such as IL-10, TGF-β, and VEGF.

Macrophages in tumors, also called tumor-related macrophages (TAMs), often express many genes typical of the M2 phenotype. Chemical attractants secreted by tumors and stromal cells recruit monocytes into tumor tissue, driving them to differentiate into M2 tumor-associated macrophages, thereby supporting tumor growth and metastasis.

After administration of the recombinant bacterium of the invention to the tumor, the recombinant bacterium produces and releases a therapeutic protein (s) into the tumor environment.

Preferably, the released therapeutic protein (s) enhance and drive these immune cells in the direction of the _TH1 immune response, which in turn reduces the production of vascular endothelial growth factor (VEGF). Bring.

Studies indicate that expression of VEGF and its receptors on macrophages plays a role in regulating the immune system and its surrounding microenvironment. In particular, M2 tumor-related macrophages actually express large amounts of VEGF and support angiogenesis and immunoregulation.

It has also been described that VEGF inhibits dendritic cell differentiation. In contrast, further, VEGF has also been shown to inhibit T cell development, thereby contributing to tumor-induced immunosuppression.

Reduced production of VEGF preferably further supports antitumor effects by preventing immunosuppression and angiogenesis.

In a preferred embodiment, the recombinant bacterium according to claim 1 is preferably a cell receptor activator, eg, a glucocorticoid-inducible TNFR-related protein (GITR), a tumor necrosis factor receptor super, also known as 4-1BB. Family member 9 (TNFRSF9), differentiation antigen group 40 (CD40), tumor necrosis factor receptor superfamily member 4 (TNFRSF4), also known as CD134 or OX40 receptor, and combinations thereof, costimulatory receptor activation. Factors, immune checkpoint inhibitors such as cytotoxic T lymphocyte-related protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-1L), programmed death ligand 2 ( PD-1L2) and their combinations, chimeric antigen receptors such as CAR-T and their combinations, prodrug activators such as HSV-tk, citidine deaminase and their combinations, cytokines, chemokines, growth factors. , Decoy receptor ligands, antibodies, soluble receptors, decoy receptors, and combinations thereof, preferably granulocytes-macrophages-colony stimulating factor (GM-CSF), interferon alpha, preferably interferon alpha 2 (IFNA2), interferon. Beta, interferon gamma, granulocyte-colony stimulating factor (G-CSF), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin- 8 (IL-8), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin- At least one selected from the group consisting of 21 (IL-21), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-23 (IL-32), and combinations thereof. Includes at least one nucleic acid sequence encoding one heterologous factor.

SEQ ID NO: 1 corresponds to the nucleic acid sequence of the expression plasmid pAUC1010, which is also shown in FIG. SEQ ID NO: 2 corresponds to the nucleic acid sequence of the PgadC promoter used in the Examples, which is also shown in FIG. 2b. SEQ ID NO: 3 is L.I. Corresponds to the amino acid sequence of the Usp45 signal sequence of Lactis, which is also shown in FIG. SEQ ID NO: 4 is L.I. Corresponds to the 3'end of the Lactis 16S RNA, which is also shown in FIG.

SEQ ID NO: 5 corresponds to the amino acid sequence of the mature form of hFGF-2-155, which is also shown in FIG. 5a. SEQ ID NO: 6 corresponds to the amino acid sequence of the hFGF-2-153 variant used in the Examples, which is also shown in FIG. 5b. SEQ ID NO: 7 corresponds to the amino acid sequence of the recombinant hFGF-2-153 precursor protein used in the Examples, which is also shown in FIG. 5c.

SEQ ID NO: 8 corresponds to the amino acid sequence of the mature form of hIL-4, which is also shown in FIG. 6a. SEQ ID NO: 9 corresponds to the amino acid sequence of the hIL-4 variant used in the Examples, which is also shown in FIG. 6b. SEQ ID NO: 10 corresponds to the amino acid sequence of the recombinant hIL-4 precursor protein used in the Examples, which is also shown in FIG. 6c.

SEQ ID NO: 11 corresponds to the amino acid sequence of the mature form of hCSF1, which is also shown in FIG. 7a. SEQ ID NO: 12 corresponds to the amino acid sequence of the hCSF1 variant used in the examples, which is also shown in FIG. 7b. SEQ ID NO: 13 corresponds to the amino acid sequence of the recombinant hCSF1 precursor protein used in the Examples, which is also shown in FIG. 7c.

SEQ ID NO: 14 corresponds to the nucleic acid sequence of the synthetic CFI construct used in the Examples, which is also shown in FIG. 8b. SEQ ID NO: 15 corresponds to the nucleic acid sequence of the expression plasmid pC-CFI used in the Examples, which is also shown in FIG. 9b.

SEQ ID NO: 16 corresponds to the amino acid sequence of the mature form of mIL-18, which is also shown in FIG. 10a. SEQ ID NO: 17 corresponds to the amino acid sequence of the mIL-18 variant used in the Examples, which is also shown in FIG. 10b. SEQ ID NO: 18 corresponds to the amino acid sequence of the recombinant mIL-18 precursor protein used in the Examples, which is also shown in FIG. 10c.

SEQ ID NO: 19 corresponds to the amino acid sequence of the mature form of mGM-CSF, which is also shown in FIG. 10a. SEQ ID NO: 20 corresponds to the amino acid sequence of the mGM-CSF variant used in the Examples, which is also shown in FIG. 10b. SEQ ID NO: 21 corresponds to the amino acid sequence of the recombinant mGM-CSF precursor protein used in the Examples, which is also shown in FIG. 10c.

SEQ ID NO: 22 corresponds to the nucleic acid sequence of the synthetic mEG construct used in the Examples, which is also shown in FIG. 12b. SEQ ID NO: 23 corresponds to the nucleic acid sequence of the expression plasmid pC-mEG used in the Examples, which is also shown in FIG. 12c.

SEQ ID NO: 24 corresponds to the amino acid sequence of the mature form of mIL-12 beta, which is also shown in FIG. 13a. SEQ ID NO: 25 corresponds to the amino acid sequence of the mature form of mIL-12 alpha isoform 2, which is also shown in FIG. 13b. SEQ ID NO: 26 corresponds to the amino acid sequence of the mature form of the recombinant mouse interleukin-12 fusion protein used in the Examples, which is also shown in FIG. 13c. SEQ ID NO: 27 corresponds to the amino acid sequence of the recombinant mouse interleukin-12 precursor protein used in the Examples, which is also shown in FIG. 13d.

SEQ ID NO: 28 corresponds to the amino acid sequence of the mature form of mouse interferon alpha-2, which is also shown in FIG. 14a. SEQ ID NO: 29 corresponds to the amino acid sequence of the mouse interferon alpha-2 variant used in the examples, which is also shown in FIG. 14b. SEQ ID NO: 30 corresponds to the amino acid sequence of the recombinant mouse interferon alpha-2 precursor protein used in the Examples, which is also shown in FIG. 14c.

SEQ ID NO: 31 corresponds to the nucleic acid sequence of the synthetic mTEA construct used in the Examples, which is also shown in FIG. 15a. SEQ ID NO: 32 corresponds to the nucleic acid sequence of the expression plasmid pC-mTEA used in the Examples, which is also shown in FIG. 15c.

SEQ ID NO: 33 corresponds to the nucleic acid sequence of the synthetic mGTE construct used in the Examples, which is also shown in FIG. 16a. SEQ ID NO: 34 corresponds to the nucleic acid sequence of the expression plasmid pC-mGTE used in the Examples, which is also shown in FIG. 16c.

SEQ ID NO: 35 corresponds to the nucleic acid sequence of the synthetic mCherry construct used in the Examples, which is also shown in FIG. 17b. SEQ ID NO: 36 corresponds to the nucleic acid sequence of the expression plasmid pC-mCherry used in the Examples, which is also shown in FIG. 18b.

The following figures and examples are given for illustration purposes only. The present invention should not be construed as being limited to the following examples.

figure:
In the figure, the following abbreviations are used: "all" refers to the alanin plasmid gene used to grow the host strain in the absence of d-alanine; "T" refers to the terminator sequence; "-35" And "-10" refer to the -35 and -10 elements of the promoter, respectively; "IR" refers to the reverse repeat sequence; "repC" and "repA" refer to the prokaryotic system required for plasmid replication by bacterial cells. A gene, where "gadR" refers to the gadR gene and "RBS" is the ribosome binding site of the indicated gene, eg, the ATP synthase subunit gamma (atpG) gene and / or the galactosid O-acetyltransferase (lacA) gene. Refers to; "ssUsp45" refers to the Usp45 secretory signal.

FIG. 1a shows a plasmid map of the expression plasmid pAUC1010. The corresponding nucleic acid sequence is shown in FIG. 1b.
FIG. 2a shows a schematic diagram of a control element comprising the chloride-inducible promoter PgadC. The corresponding nucleic acid sequence is shown in FIG. 2b, which includes the regulatory element containing the PgadC promoter region, the gadR gene, and the ribosome binding site (RBS) and start codon of the gadC gene.
FIG. 3 shows the amino acid sequence of the secretory signal of the Lactococcus protein Usp45.
FIG. 4 shows L. The nucleic acid sequence of the 3'end of 16S rRNA of Lactis is shown.
FIG. 5a shows the amino acid sequence of the mature form of human FGF-2-155. The amino acid sequence of the variant of hFGF2-153 used in the examples is shown in FIG. 5b. The amino acid sequence of the recombinant hFGF-2-153 precursor protein used in the examples is shown in FIG. 5c.
FIG. 6a shows the amino acid sequence of the mature form of human IL-4. The amino acid sequence of the hIL-4 variant used in the examples is illustrated in FIG. 6b. The amino acid sequence of the recombinant hIL-4 precursor protein used in the examples is shown in FIG. 6c.
FIG. 7a shows the amino acid sequence of the mature form of human CSF-1. The amino acid sequence of the hCSF-1 variant used in the examples is shown in FIG. 7b. The amino acid sequence of the recombinant hCSF-1 precursor protein used in the examples is shown in FIG. 7c.
FIG. 8a shows a schematic diagram of the de novo-synthesized CFI construct used in the examples. The corresponding nucleic acid sequence of the de novo synthesized CFI construct is shown in FIG. 8b. The nucleic acid sequence of the expression plasmid called pC-CFI is shown in FIG. 9b. A schematic diagram of the expression plasmid pC-CFI is illustrated in FIG. 9a.
FIG. 10a shows the amino acid sequence of the mature form of mouse IL-18. The amino acid sequence of the variant of mIL-18 used in the examples is shown in FIG. 10b. The amino acid sequence of the recombinant mIL-18 precursor protein used in the examples is shown in FIG. 10c.
FIG. 11a shows the amino acid sequence of the mature form of mouse GM-CSF. The amino acid sequence of the variant of mGM-CSF used in the examples is shown in FIG. 11b. The amino acid sequence of the recombinant mGM-CSF precursor protein used in the examples is shown in FIG. 11c.
FIG. 12a shows the nucleic acid sequence of the synthetic mEG construct used in the Examples. The nucleic acid sequence of the expression plasmid, called pC-mEG, is shown in FIG. 12c, and a schematic diagram of the corresponding expression plasmid pC-mEG used in the Examples is shown in FIG. 12b.
FIG. 13a shows the amino acid sequence of the mature form of the mouse IL-12 subunit beta. The amino acid sequence of the mature interleukin-12 subunit alpha isoform 2 is shown in FIG. 13b. The amino acid sequence of the mature form of the recombinant interleukin-12 fusion protein used in the examples is shown in FIG. 13c. The amino acid sequence of the recombinant mIL-12 precursor protein used in the examples is shown in FIG. 13d.
FIG. 14a shows the amino acid sequence of mature mouse IFNa2. The amino acid sequence of the variant of mIFNa2 used in the examples is shown in FIG. 14b. The amino acid sequence of the synthetic miFNa2 precursor protein used in the examples is shown in FIG. 14c.
FIG. 15a shows the nucleic acid sequence of the synthetic mTEA construct used in the examples. The nucleic acid sequence of the expression plasmid called pC-mTEA is shown in FIG. 15c. A schematic diagram of the expression plasmid pC-mTEA is illustrated in FIG. 15b.
FIG. 16a shows the nucleic acid sequence of the synthetic mGTE construct used in the examples. The nucleic acid sequence of the expression plasmid called pC-mGTE is shown in FIG. 16c. A schematic diagram of the corresponding expression plasmid pC-mEG used in the Examples is illustrated in FIG. 16b.
FIG. 17a shows a schematic diagram of the synthetic mCherry construct used in the examples. The corresponding nucleic acid sequence of the synthetic mCherry construct is shown in FIG. 17b.
The nucleic acid sequence of the expression plasmid called pC-mCherry used in the examples is shown in FIG. 18b. A schematic diagram of the expression plasmid pC-mCherry is illustrated in FIG. 18a.
FIG. 19a shows L. A comparison of the growth curves of Lactis NZ1330 (pC-mCherry), after induction of mCherry gene expression by 100 mM NaCl (“L. Lactis NZ1330 :: pC-mCherry + NaCl”) and without induction (“L. Lactis NZ1330:”). : PC-mCherry + mq "), respectively, determined by measuring the optical density (OD600) at a wavelength of 600 nm at the indicated time point (T) in Example 3, respectively.
FIG. 19b shows L. Comparison of mCherry fluorescence obtained from Lactis NZ1330 (pC-mCherry), after induction of mCherry gene expression with 100 mM NaCl ("NZ1330 :: pC-mCherry + NaCl") and without induction ("NZ1330 :: pC-"). The comparison of mCherry + MQ ") is shown in artificial units and was measured at the time indicated by the minute under the conditions described in Example 3.
FIG. 20a shows a Western blot analysis of a recombinant bacterium called AUP1602-C expressing human CSF-1, human FGF-2, and human IL-4 from the expression plasmid pC-CFI obtained in Example 1.4. ing. "Anti-CSF-1", "Anti-FGF-2", and "Anti-IL-4" are described in Example 3 to detect the expression of human CSF-1, human FGF-2, and human IL-4, respectively. Refers to the primary antibody used.
20b and 20c are Western blots of a recombinant bacterium called AUP5563-C expressing mouse IL-12, mouse IL-18, and mouse GM-CSF from the expression plasmid pC-mGTE obtained in Example 1.7. Shows the analysis. "Anti-GM-CSF" and "anti-IL-18" refer to the primary antibody used in Example 3 to detect expression of mouse GM-CSF and mouse IL-18, respectively.
FIG. 20d shows AUP555m-C expressing mouse IL-12, mouse IL-18, and mouse IFNa from the expression plasmid pC-mTEA obtained in Example 1.6, as well as mouse IL-12, mouse IL-18, and mouse. A Western blot analysis of a recombinant bacterium called AUP5563-C expressing GM-CSF from the expression plasmid pC-mGTE obtained in Example 1.7 is shown. "Anti-IL-12" refers to the primary antibody used in Example 3 to detect the expression of mouse IL-12.
FIG. 21 shows recombinant L. cerevisiae containing an AUC1000 (pC-mCherry) construct under the conditions described in Example 4. Lactis (“PGAD-mCherry”) and control bacteria without an expression plasmid (“L. lactis (control)”), i. t. Fluorescence imaging of BALB / c mice induced tumors 48 hours after injection is shown. The blue circle points to the location of the tumor.
FIG. 22 shows the average percentage wound area of all treatment groups in the wound closure experiment described in Example 5.
FIG. 23 shows the average percentage wound contraction of all treatment groups in the wound closure experiment described in Example 5.
FIG. 24 shows the percentage of responded wounds in each treatment group on day 1 of the wound closure experiment described in Example 5.
25a-27b are described in Example 6 of the detection of human FGF-2, human IL-4, and human CSF-1 in the wound fluid of mice treated in the wound closure experiment described in Example 5. The result is shown.
28a-28c show Example 7 of specific tumor volumes of vehicle-treated control mice, mice treated with AUP5563-C4, and anti-m-CTLA-4 treated control mice at the indicated time points. It shows the result that has been done.
FIG. 29 shows C57BL76 mice treated in Example 8 with the respective combinations of the drug products AUP2059 (mIL18 / mGM-CSF) and AUP5551-C (mIL12 / mIL18 / mIFNa2b) obtained in Example 3, and a vehicle-treated control. It shows the survival curve of mice.

example:
I) General experimental procedure:
Unless otherwise claimed, the examples were carried out following the protocol of the manufacturer of the analytical system. Unless otherwise claimed, the indicated chemicals are Sigma-Aldrich Chemie GmbH (Munich, DE), Merck KGaA (Darmstadt, DE), Thermo Fisher Scientific Inc. (Waltham, MA, US), or commercially obtained from Becton, Dickinson and Company (Franklin Lakes, NJ, US).

I. 1 Growth medium For different purposes, cells were grown in different media.

For the general cloning procedure, M17 medium (Oxoid Deutschland GmbH, Wesel, DE) containing 1 wt% glucose or lactose, respectively, was used.

E. The coli strain was grown on TY medium. The recipe for the TY medium used is as follows:
1 wt% Tryptone 0.5 wt% Yeast Extract 0.5 wt% NaCl

IM1 medium was used for fermentation and other functional growth experiments, but it is free of animal-derived components. The only remaining animal-derived ingredient lactose can be obtained in pharmaceutical quality.

The recipe for the IM1 medium used is as follows:
1 wt% lactose 2 wt% β-Na glycerophosphate
1.5 wt% soybean peptone 1 wt% yeast extract 1 mM ו ₄ × 7H ₂ O
0.1 mM MnSO ₄ × 4H ₂ O
pH 6.7
Sterilization: 110 ° C, 15 min

No buffer (eg, Na β-glycerophosphate) is added during fermentation, because the pH was automatically controlled with 2.5 M NaOH. This buffer neutralizes the lactic acid produced and allows the culture to reach a cell density of OD ₆₀₀ = 10-15.

Stock L. For the growth of Lactis NZ1330 and NZ9130, D-alanine was added to the medium at a final concentration of 200 μg / mL.

I. 2 Bacterial strains The following commercial bacterial strains were used. Lactococcus lactis strains NZ3900 and NZ1330 were obtained from MoBiTec GmbH (Göttingen, DE).

L. The lactis strain NZ1330 contains a gene encoding the deleted alanine racemase (allr) (Δallr). Deletion of the allr gene resulted in the auxotrophy of the essential component D-alanine. Unless the allr gene is provided on a plasmid, each strain cannot grow on medium without D-alanine.

I. 3 Molecular Cloning Technology For molecular cloning, for example, Green and Sambrook (2012): “Molecular cloning: a laboratory manual”, fourth edition, Cold Spring Harbor Laboratory Description (Col. Standard techniques were used.

Different synthetic DNA constructs were produced by BaseClear (Leiden, NL). The constructs are the purified plasmid and E.I. Obtained as a clone of Kori.

Plasmid DNA was prepared using standard DNA isolation from Birnboim, H. et al. C. and Dolly, J. et al. Isolated according to (1979). Synthetic gene constructs were excised from those plasmids using selected restriction enzymes, purified and ligated to their respective target plasmids. Ligase was performed using the Anza T4 ligase master mix (Thermo Fisher Scientific Inc.) according to the supplied protocol.

A different ligation mixture was selected from L. The lactis strain (L. lactis NZ1330 or L. lactis NZ9130) was transformed by electroporation and plated on a suitable medium.

I. Preparation of 4 strains of electro-competent cells and electroporation Electro-competent cells were prepared according to the following protocol. L. Lactis NZ1330 and NZ9130 were grown overnight at 30 ° C. with 10 mL SMGG medium, which was used to inoculate 100 mL SMGG medium.

Cells were grown until they reached an OD of ₆₀₀ of ~ 0.5. The cells were washed 3 times with ice-cold wash buffer and resuspended in 1 mL wash buffer. 40 μL aliquots were stored at −80 ° C. Cellular electrocompetent properties were tested using standard electroporation procedures.

To this goal, 40 μL of cells are thawed on ice and mixed with 0.5 μL of plasmid DNA in an ice-cold electroporation cuvette, 2500 volt, 25 μF, 200 ohm Eppendorf® electroporator (registered trademark). It was applied using Eppendorf AG, Hamburg, DE).

The cells were resuspended in 4 mL of SMG17MC medium and incubated at 30 ° C. for 2 hours. Different amounts of culture were plated on GSM17 agar and incubated at 30 ° C.

Buffer solution used:
SMGG medium: Commercially available M17 medium + 0.5 M sucrose + 0.5 wt% glucose + 1 wt% glycine SMG17MC medium: Commercially available M17 medium + 0.5 M sucrose + 0.5 wt% glucose + 20 mM MgCl ₂
+ 2 mM CaCl ₂
GSM17 agar: M17 agar obtained from Oxoid Germany GmbH (Wesel, DE) + 0.5 M sucrose + 0.5 wt% glucose wash buffer: 0.5 M sucrose + 10 wt% glycerol

I. 5 Selection of transformants The transformants are transferred from plate to new plate and into tubes by 3 ml of medium M17 (supplemented with 1% glucose or lactose) and incubated at 30 ° C., with the DNA on I. .. Isolated as outlined in Section 3. Clones were screened for the presence and orientation of inserts using selected restriction endonucleases and agarose gel electrophoresis.

Positive clones were selected, cultured and stored at -80 ° C.

I. 6 Culture and induction of gene expression Selected clones were inoculated into IM1 medium supplemented with 0.5% glucose or lactose and grown overnight at 30 ° C. At t = 0, the culture was inoculated 1: 100 into 45 mL of medium and incubated. At OD ₆₀₀ = 0.5, the cultures were divided into three separate 15 ml cultures, which were induced by the addition of NaCl (0 mM, 100 mM, or 500 mM).

After 3 hours of gene induction, cells and supernatant were separated by centrifugation (10 min at 6,000 rpm).

Prior to further treatment, cells were frozen at −20 ° C. The supernatant was mixed with a third volume of TCA (see below) and stored at -20 ° C.

I. 7 Cell-free extract preparation and trichloroacetic acid (TCA) precipitation Cell-free extract was prepared by bead beating according to standard working protocols. The procedure is based on the use of 50-100 μm glass beads. The sample was kept on ice as much as possible to prevent proteolytic disintegration of the protein. After the final centrifugation step, cell-free extract was collected and stored at −20 ° C.

TCA precipitation was performed by adding 1 volume TCA to 4 volumes of culture supernatant. The mixture was incubated at −20 ° C. until further use. To obtain TCA-precipitated protein, a 1 mL sample was taken and centrifuged at 14,000 rpm for 10 min. The supernatant was tilted and the pellet was dried by stove at 65-70 ° C. Later, SDS-PAGE sample buffer containing 2% β-mercaptoethanol was added and the sample was denatured by incubation at 100 ° C. for 10 min.

I. 8 Dodecyl sodium polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting SDS-PAGE (15 wt% running gel) are essentially Laemmli, U.S.A. K. It was done according to (1970). After electrophoresis, the gel is stained with Coomassie R-250 (Bio-Rad Laboratories, Inc., Hercules, CA, US) or the protein is semi-driving rolling system (Owl®, Thermo Fisher Scientific, US). By means of Inc.), they were transferred to a membrane of polyvinylidene fluoride (PVDF) or nitrocellulose according to the manufacturer's instructions.

The PVDF or nitrocellulose membrane contains blocking with Tris buffered saline containing 0.05 wt% Tween 20 (TBST) containing 2 wt% bovine serum albumin (BSA), followed by 1 wt% BSA over 30 minutes. It was treated by incubation with specific primary antibodies produced for each protein diluted by TBST.

After that, the blots were washed 3 times with TBST for 10 minutes. After washing, for example, Santa Cruz Biotechnology, Inc. Alkaline phosphatase-conjugated anti-rabbit secondary antibody (Dallas, TX, US, catalog nr. Sc-2004) derived from goat was applied (1: 7500 in TBST + 1 wt% BSA). Unbound antibody was washed off with TBST (3 × 10 min.) And then enzymatically stained according to standard procedures with NBT and BCIP.

After sufficient color development, the reaction was terminated by rinsing with water and the membrane was dried and photographed or scanned.

Alternatively, LI-COR, Inc. conjugated with a near infrared (NIR) fluorescent dye. Donkey anti-goat antibody IRDye® 680RD from (Lincoln, NE, US) was used according to the manufacturer's protocol. Detection of bound secondary antibody is performed by LI-COR, Inc. It was performed according to the manufacturer's protocol using the Odyssey® imaging system from.

Tris buffered saline having 0.05 wt% Tween 20 is described in Carl Roth GmbH + Co. Prepared according to the manufacturer's instructions using Roti® fair TBST 7.6 tablets obtained from KG (Karlsruhe, DE).

For the detection of each recombinant protein, Abcam PLC (Cambridge, UK), Bio-Rad Laboratories Inc. (Hercules, CA, US), and LifeSpan BioSciences, Inc. The following primary antibody from (Seattle, WA, US) was used.
-Anti-FGF-2 antibody-Rabbit polyclonal antibody against FGF-2 (Abcam PLC, ab126861),
-Anti-IL-4 antibody-Rabbit polyclonal antibody against IL-4 (Abcam PLC, ab9622),
-Anti-M-CSF antibody-Rabbit polyclonal antibody against M-CSF (Abcam PLC, ab9693),
-Anti-mouse GM-CSF antibody-Rabbit polyclonal antibody against mouse GM-CSF (Bio-Rad Laboratories Inc., AAM16G),
Anti-mouse IL-18 antibody-Rabbit polyclonal antibody against mouse IL-18 (LifeSpan BioSciences, Inc., LS-C147100),
Anti-mouse IL-12 antibody-Goat anti-mouse antibody against IL-12 (Biorad Laboratories Inc., AAM33).

I. 9 Fermentation Fermentation was carried out by a 0.5 L Multifors 6 fermenter array (Inforces Benelux, Velp, NL). Inoculation was performed with 1% preculture in IM-1 medium; stirrer speed was 200 rpm and incubation temperature was 30 ° C. During operation, the pH was controlled with 2.5 M NaOH. The addition of pH, temperature, and NaOH was continuously monitored.

I. 10 Fluorescent protein measurements Strains containing constructs with the mCherry reporter gene under the control of different promoters were grown and processed as described above. The mCherry activity was measured by a Synergy HT (BioTek Instruments, Inc., Winooski, VT, US) fluorescence spectrophotometer.

Tauer et al. As described by (2014), the excitation was at 530 nm, the emission was measured at 590 nm, and a gain of 120 was applied.

Example 1 Creation of expression plasmid 1.1 Creation of plasmid pAUC1010 The plasmid pAUC1010 was prepared by L. It was used in the next experiment for the expression of heterologous genes by lactis. The plasmid pAUC1010 is based on the native rolling circle plasmid of Lactococcus lactis pSH71, deVos, W. et al. M. (1987) and de Vos, W. et al. M. and Simons, G.M. As described in (1994).

The plasmid pAUC1010 was prepared by L. It contains the alanine racemase (all) gene, which includes its terminator of lactis, as a selectable marker, which contains L. It can be used for lactis all knockout mutant strains, such as NZ1330 (Bron, PA et al. (2002), Hols, P. et al. (1999)). The plasmid also includes the artificial promoter Pcp14 (Ruhdal Jensen, P. and Hammer, K. (1998)).

Furthermore, the plasmid pAUC1010 is described in L. The terminator sequence of the lactis aminopeptidase N (pepN) gene (Tan, PST et al. (1992)) is contained downstream of the promoter and multicloning site (MCS). The terminator sequence allows the termination of transcription.

The nucleic acid sequence of the Lactococcus lactis subspecies Cremoris allr gene of alanine racemase (EC 5.1.1.1) is available, for example, at NCBI Accession No. Y18148.2.

The respective amino acid sequences of the alanine racemase of the Lactococcus lactis subspecies Cremoris can be found, for example, in UniProt accession number Q9RLU5 version 99 or NCBI reference SEQ ID NO: WP_11835506.1.

The nucleic acid sequence of pAUC1010 is provided in SEQ ID NO: 1 and FIG. 1b.

FIG. 1a shows a schematic overview of the plasmid pAUC1010. "All" refers to the alanine racemase gene used to grow the host strain in the absence of d-alanine; "T" refers to the terminator sequence; "repC" and "repA" are required for plasmid replication by bacterial cells. It is a prokaryotic gene.

1.2 Creation of chloride-inducible expression cassette In the following experiment, a chloride-inducible gene expression cassette was produced. The PgadC promoter from the Lactis subspecies Cremoris is described in L. et al. It was used to control the expression of different heterologous genes by lactis.

The PgadC promoter is described by Sanders et al. (1997) and Sanders et al. As described in (1998), it was used in combination with the activator gene gadR, as well as the ribosome binding site and start codon of the gadC gene, both located downstream of the PgadC promoter.

The promoter PgadC is regulated by the gadR protein, which is a positive regulator of Pgad. The gadR protein activates the PgadC promoter in the presence of chloride ions.

The expression of the gadR gene itself is not activated by chloride ions and is instead controlled by a constitutive promoter, which is also a ribosome binding site located upstream of the ATG start codon of the nucleic acid sequence encoding gadR ( RBS) were provided on a chloride-induced gene expression cassette used in the present invention.

The nucleic acid sequence of the PgadC promoter, which comprises the ribosome binding site and start codon of the gadR gene and the gadC gene, is the strain L. It was extracted from the genomic sequence of the Lactis subspecies Cremoris MG1363. Each genomic sequence is available, for example, at NCBI Accession No. NC_09004.1.

Nucleic acid sequences of chloride-inducible promoters comprising the PgadC promoter region, the gadR gene, and the ribosome binding site and start codon of the gadC gene are provided in SEQ ID NO: 2 and FIG. 2b. The ATG start codon of gadC is shown in FIG. 2b by underlining and in bold capital letters.

A schematic diagram of the control elements including the chloride-inducible promoter is shown in FIG. 2a.

Each target gene (s) encoding at least one protein to be expressed by the recombinant bacterium of the invention was attached to the ATG start codon of gadC. Furthermore, the genes encoding each protein to be expressed by the recombinant bacteria of the invention provided a secretory signal for protein secretion from the expression host.

The secretory signal was preferably derived from the Lactococcus protein Usp45, which may be described, for example, by van Asseldonk et al. (1993) and van Asseldonk et al. (1990). The amino acid sequence of the secretory signal of the Lactococcus protein Usp45 is shown in FIG. 3 and SEQ ID NO: 3.

There is a mechanism in the cell that ensures that this signal sequence is cleaved from the target protein after the protein is secreted. This reaction is catalyzed by a signal peptidase that has a specific preference for certain amino acids located around the cleavage site.

At least one protein to be expressed by the recombinant bacterium of the present invention was expressed as a recombinant precursor protein containing the respective secretory signals of the Lactococcus protein Usp45 at the N-terminus of each precursor protein.

The codon frequency of each gene (s) encoding at least one protein to be expressed by the recombinant bacterium of the invention is described in L. et al. It was adapted to the general codon usage frequency of Lactis.

Furthermore, if more than one protein should be expressed by the recombinant bacterium of the invention, the same Usp45 signal peptide sequence was used for each protein.

To avoid large identical nucleotide regions on closely proximal plasmids that can lead to recombination / deletion within the plasmid, the same signal peptide is described by L. It was encoded by different codons based on the codon degeneration and codon usage frequency of Lactis.

Furthermore, the web-based program SignalP4.1 (http://www.cbs.dtu.dk/services/) is used to optimize the cleavage site where the signal peptide is cleaved from the synthetic precursor protein to produce the mature protein. SignalP /) was used.

Also, if more than one protein should be expressed by the recombinant bacterium of the invention, preferably from at least one operon, each of the respective genes binds to ribosomes to improve protein expression. The site (RBS) was provided.

Preferably, the ribosome binding site of the gadC gene was used as the first gene of the operon containing nucleic acid sequences encoding two or more proteins. Other genes in the operon include endogenous L. Ribosome binding sites of the lactis gene, such as the ATP synthase subunit gamma (atpG) gene and / or the galactoside O-acetyltransferase (lacA) gene, were used.

Suitable ribosome binding sites are, for example, L. Based on a good fit with the 3'end of Lactis 16S rRNA, Bollotin et al. As described in (2001), known L. From the lactis nucleic acid sequence, for example, accession no. L.A. available from NCBI at AE005176.1. Lactis subspecies Lactis IL1403 genome or L.I. It can be selected from the nucleotide sequence of the Lactis subspecies Cremoris MG1363 genome.

L. The 3'end of the 16S rRNA of Lactis (5'GGAUCACCUCCUUUCU3') is shown in SEQ ID NO: 4 and in FIG.

Synthetic nucleic acid constructs comprising chloride-inducible promoters, and at least one nucleic acid sequence encoding at least one protein to be expressed by the recombinant bacteria of the invention, are de novo synthesized, as outlined above, and then. Each expression plasmid was ligated into a suitable strain, preferably L. Transformed into lactis.

The following expression plasmid was produced.

1.3 Creation of expression plasmids for expression of mCherry Different constructs containing the mCherry gene were prepared under the control of PgadC. mCherry is described by Beilharz K, et al. It is the fluorescent protein first described by (2015).

The nucleic acid sequence encoding the mCherry protein is available at NCBI Accession No. KJ90810.1. The corresponding amino acid sequence is available at NCBI Accession No. AIL2875.9.1.

The mCherry coding sequence was fused to the PgadC promoter. A schematic diagram of each insert is shown in FIG. 17a.

The nucleic acid sequence of the synthetic mCherry construct is shown in FIG. 17b and SEQ ID NO: 33. A schematic diagram of the synthetic mCherry construct is shown in FIG. 17a.

The completed gene synthesis product is E.I. It was obtained from BaseClear as a fragment cloned on the coli vector pUC57.

The pUC57 plasmid with each gene synthesis product was digested with restriction enzymes SphI and BglII. The SphI and BglII fragments containing the respective gene synthesis products were isolated by phenol extraction and ethanol precipitation and ligated into the SphI and BglII cut plasmid pAUC1010, resulting in the production of an expression plasmid called pC-mCherry. ..

The nucleic acid sequence of the expression plasmid, called pC-mCherry, is shown in FIG. 18b and SEQ ID NO: 34. A schematic diagram of the expression plasmid pC-mCherry is illustrated in FIG. 18a.

1.4 Creation of expression plasmids for expression of hFGF-2, hIL-4, and hCSF-1 from a single operon Human fibroblast growth factor 2 (hFGF-2), human interleukin 4 (hIL- 4) and human colony stimulating factor 1 (hCSF-1) were added to L. The following nucleic acid sequences were generated for expression by Lactis NZ1330.

14.1 Human fibroblast growth factor 2 (hFGF-2)
The amino acid sequence used for the expression of hFGF-2 is derived from the 288 amino acid sequence of the human FGF-2 precursor available at NCBI Accession No. NP_001997.5.

The mature form of hFGF-2 contains 155 amino acids and is subsequently referred to as hFGF-2-155. It has a molecular weight of 17.3 kDa and a pI of 9.85. The molecule contains four cysteine residues.

L. The hFGF-2 variant to be expressed by lactis lacks the first two amino acids methionine and alanine and contains 153 amino acids. Therefore, this variant was subsequently referred to as hFGF-2-153. hFGF-2-153 has a molecular weight of 17.1 kDa and a pI of 9.85. This variant contains 4 cysteine residues.

The corresponding amino acid sequences of human FGF-2-155 are shown in FIG. 5a and SEQ ID NO: 5. The underlined amino acids shown in FIG. 5a refer to the first two amino acids, methionine and alanine, which are missing in variant hFGF2-153. The amino acid sequence of the variant of hFGF2-153 is shown in FIG. 5b and in SEQ ID NO: 6.

The sequence of FGF-2-155 is the post-secretory mature human FGF-2 sequence. In vivo, this sequence is further processed. However, the various existing recombinants use this 155 amino acid sequence with or without an N-terminal methionine residue.

To express and secrete hFGF2, for example, in the supernatant of growth medium, a signal peptide was added to the N-terminus as described above to produce recombinant hFGF-2-153 precursor protein.

The respective amino acid sequences of the recombinant hFGF-2-153 precursor protein are shown in FIG. 5c and SEQ ID NO: 7. The underlined amino acids shown in FIG. 5c refer to the secretory signal of the Lactococcus protein Usp45.

1.4.2 Human interleukin-4 (hIL-4)
The mature protein of hIL-4 contains 129 amino acids, which correspond to amino acids 25-153 of the amino acid sequence available at NCBI Accession No. NP_0005800.1. The mature protein has a molecular weight of 14.96 kDa and a pI of 9.36. The molecule contains 6 cysteine residues.

The amino acid sequence of mature hIL-4 is shown in FIG. 6a and SEQ ID NO: 8.

L. The hIL-4 variant to be expressed by lactis contains an additional alanine residue at the N-terminus of the mature protein and therefore contains 130 amino acids. It has a molecular weight of 15.03 kDa and a pI of 9.36. This variant also contains 6 cysteine residues.

The corresponding amino acid sequences of the human hIL-4 variant to be expressed are shown in FIG. 6b and SEQ ID NO: 9. The underlined amino acids shown in FIG. 6b refer to the additional alanine residue at the N-terminus of the amino acid sequence.

To express and secrete hIL4, for example, in the supernatant of the growth medium, a signal peptide was added to the N-terminus as described above to produce a recombinant hIL-4 precursor protein. The respective amino acid sequences of the recombinant hIL-4 precursor protein are shown in FIG. 6c and SEQ ID NO: 10. The underlined amino acids shown in FIG. 6c refer to the secretory signal of the Lactococcus protein Usp45.

1.4.3 Human colony stimulating factor 1 (hCSF1)
The amino acid sequence of hCSF1 used for expression is derived from the 554 amino acid sequence of human CSF1 precursor isoform a, which is available at NCBI Accession No. NP_000748.3.

The mature form of hCSF1 spans the precursor amino acids 33-450. The amino acid sequence is shown in FIG. 7a and SEQ ID NO: 11.

The amino acid sequence to be expressed begins with amino acid 33 (E), the first amino acid of mature hCSF1 and ends with amino acid 181 (Q).

Furthermore, an additional 9 amino acids occupying positions 480-488 of the native protein were added to the C-terminus (GHERQSEGS). An additional alanine residue was added to the N-terminus to improve the removal of the signal peptide by signal peptidase. The resulting amino acid sequence is shown in FIG. 7b and SEQ ID NO: 12.

The additional alanine residue at the N-terminus of FIG. 7b is underlined. The additional 9 amino acids added to the C-terminus are shown in bold in FIG. 7b. The protein encoded by this sequence contains 159 amino acids and has a molecular weight of 18.5 kDa and a pI of 4.72. The protein contains 7 cysteine residues.

To express and secrete hCSF1, for example in the supernatant of growth medium, a signal peptide was added to the N-terminus as described above to produce a recombinant hCSF1 precursor protein. The respective amino acid sequences of the synthetic hCSF1 precursor protein are shown in FIG. 7c and SEQ ID NO: 13. The underlined amino acids shown in FIG. 7c refer to the secretory signal of the Lactococcus protein Usp45.

1.4.4 Production of expression constructs The synthetic hFGF-2-153 precursor protein shown in Figure 5c and SEQ ID NO: 7, the synthetic hIL-4 precursor protein shown in Figure 6c and SEQ ID NO: 10, and. The amino acid sequence of the synthetic hCSF1 precursor protein shown in FIG. 7c and SEQ ID NO: 13 was translated into the corresponding nucleic acid sequence, whereby the codon usage frequency was L. It was adapted to the general codon usage frequency of Lactis.

Two constructs were made by two of the six sequences of three genes (FGF2, IL4, CSF1):
a) hCSF1, hFGF2, hIL4 (CFI)
b) hIL4, hCSF1, hFGF2 (ICF)

For the design of the operon, each gene was provided with its own ribosome binding site for initiation of translation and its own signal peptide for protein secretion. The same signal sequence (ssUsp45) encoded by different codons based on the codon decompression and codon frequency of lactis was used.

The ribosome binding site of the gadC gene was used for each of the first genes of the two constructs. The ribosome binding sites of the ATP synthase subunit gamma (atpG) gene and the galactoside O-acetyltransferase (lacA) gene were used for the other two genes of each of the two constructs.

The nucleic acid sequence of the de novo-synthesized CFI construct is shown in FIG. 8b and SEQ ID NO: 14. A schematic diagram of the de novo synthesized CFI construct is illustrated in FIG. 8a.

The completed gene synthesis products are each E.I. It was obtained from BaseClear as a fragment cloned on the coli vector pUC57.

The pUC57 plasmids with the respective gene synthesis products were digested with the restriction enzymes SphI and BglII from New England Biolabs (Ipswich, MA, US), respectively. The SphI and BglII fragments containing the respective gene synthesis products were isolated by phenol extraction and ethanol precipitation and ligated into the SphI and BglII cut plasmid pAUC1010, resulting in the production of an expression plasmid called pC-CFI. ..

The nucleic acid sequence of the expression plasmid, called pC-CFI, is shown in FIG. 9b and SEQ ID NO: 15. A schematic diagram of the expression plasmid pC-CFI is illustrated in FIG. 9a.

1.5 Creation of expression plasmids for the expression of mIL-18 and mGM-CSF from a single operon Mouse interleukin 18 (mIL18) and mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) were prepared by L. The following nucleic acid sequences were generated for expression by Lactis NZ1330.

1.5.1 Mouse interleukin-18 (mIL-18)
The mature protein of mIL-18 contains 157 amino acids, which correspond to amino acids 36-192 of the amino acid sequence of the mouse interleukin-18 isoform a precursor available at NCBI Accession No. NP_032386.1.

The amino acid sequence of mature mouse IL-18 is shown in FIG. 10a and SEQ ID NO: 16.

L. The mIL-18 variant, which should be expressed by lactis, contains an additional alanine residue at the N-terminus of the mature protein. The corresponding amino acid sequences of the mouse IL-18 variant to be expressed are shown in FIG. 10b and SEQ ID NO: 17. The underlined amino acids shown in FIG. 10b refer to the additional alanine residue at the N-terminus of the amino acid sequence.

To express and secrete mIL-18, for example in the supernatant of growth medium, a signal peptide was added to the N-terminus as described above to produce a synthetic mIL-18 precursor protein. The respective amino acid sequences of the synthetic mIL-18 precursor protein are shown in FIG. 10c and SEQ ID NO: 18. The underlined amino acids shown in FIG. 10c refer to the secretory signal of the Lactococcus protein Usp45.

1.5.2 Mouse Granulocyte-Macrophage Colony Stimulating Factor (mGM-CSF)
Mature mGM-CSF contains 129 amino acids, which correspond to amino acids 18-141 of the amino acid sequence of the mouse granulocyte-macrophage colony-stimulating factor precursor available in the NCBI reference sequence: NP_030999.2.

The amino acid sequence of mature mouse GM-CSF is shown in FIG. 11a and SEQ ID NO: 19.

L. The mGM-CSF variant that should be expressed by lactis contains an additional alanine residue at the N-terminus of the mature protein, which replaces the N-terminus serine to optimize cleavage of the signal peptide. The corresponding amino acid sequences of the mouse GM-CSF variants to be expressed are shown in FIG. 11b and SEQ ID NO: 20. The underlined amino acids shown in FIG. 11b refer to the additional alanine residue at the N-terminus of the amino acid sequence.

To express and secrete mGM-CSF, for example in the supernatant of growth medium, a signal peptide was added to the N-terminus as described above to create a recombinant mGM-CSF precursor protein. The respective amino acid sequences of the synthetic mGM-CSF precursor protein are shown in FIG. 11c and SEQ ID NO: 21. The underlined amino acids shown in FIG. 11c refer to the secretory signal of the Lactococcus protein Usp45.

1.5.3 Creation of Expression Constructs The amino acid sequences of the recombinant mIL-18 precursor protein shown in Figure 10c and SEQ ID NO: 18 and the recombinant mGM-CSF precursor protein shown in FIG. 11c and SEQ ID NO: 21 , Translated into the corresponding nucleic acid sequence, whereby the codon usage frequency is L. It was adapted to the general codon usage frequency of Lactis.

For the design of the operon, each of the two genes encoding each precursor protein was provided with its own ribosome binding site for translation initiation and its own signal peptide for protein secretion. , As outlined above, L. The same signal sequence (ssUsp45) encoded by different codons based on the codon decompression and codon frequency of lactis was used.

The ribosome binding site of the gadC gene was used for the first gene of the operon (mIL-18). For the second gene (mGM-CSF), the ribosome binding site of the ATP synthase subunit gamma (atpG) gene was used.

The nucleic acid sequence of the synthetic mEG construct is shown in FIG. 12a and SEQ ID NO: 22.

The completed gene synthesis product is E.I. It was obtained from BaseClear as a fragment cloned on the coli vector pUC57.

The pUC57 plasmids with the respective gene synthesis products were digested with the restriction enzymes SphI and BglII from New England Biolabs (Ipswich, MA, US), respectively. The SphI and BglII fragments containing the respective gene synthesis products were isolated by phenol extraction and ethanol precipitation and ligated into the SphI and BglII cut plasmid pAUC1010, resulting in the production of an expression plasmid called pC-mEG. ..

The nucleic acid sequence of the expression plasmid, called pC-mEG, is shown in FIG. 12c and SEQ ID NO: 23. A schematic diagram of the expression plasmid pC-mEG is illustrated in FIG. 12b.

1.6 Creation of expression plasmids for expression of mIL-12, mIL-18, and mIFNa from a single operon Mouse interleukin 12 (mIL-12), mouse interleukin 18 (mIL-18), and mice Interferon alpha-2 (mIFNa) was added to L. The following nucleic acid sequences were generated for expression by Lactis NZ1330.

1.6.1 Mouse interleukin-12 (mIL-12)
Interleukin-12 (IL-12) is a heterodimer cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40).

L. The mIL-12 variants that should be expressed by lactis are the mature interleukin-12 subunit beta, which forms the N-terminus of the fusion protein, and the mature interleukin-12 subunit alpha, which forms the C-terminus of the fusion protein. Designed as a fusion protein with, both were separated by a peptide linker.

The mature proteins of the mouse interleukin-12 subunit beta contain 313 amino acids, which are from amino acids 23 to 335 of the amino acid sequence of the mouse interleukin-12 subunit beta precursor available at NCBI accession number NP_001290173.1. handle.

The amino acid sequence of the mature interleukin-12 subunit beta is shown in FIG. 13a and SEQ ID NO: 24.

Mouse interleukin 12 subunit alpha exists as at least two isoforms 1 and 2. The encoded isoform 2 has a shorter N-terminus compared to isoform 1.

The amino acid sequence of the mouse interleukin 12 subunit alpha isoform 1 precursor is available at NCBI Accession No. NP_001152896.1.

The mature protein of the mouse interleukin-12 subunit alpha isoform 2 whose amino acid sequence was used for expression in the following example contains 199 amino acids, which are available at NCBI Accession No. NP_032377.1. Corresponds to amino acids 23-215 in the amino acid sequence of the Leukin 12 subunit alpha isoform 2 precursor.

The amino acid sequence of the mature interleukin-12 subunit alpha isoform 2 is shown in FIG. 13b and SEQ ID NO: 25.

The amino acid sequence of the mature form of the recombinant interleukin-12 fusion protein is shown in FIG. 13c and SEQ ID NO: 26. Furthermore, two additional amino acids (alanine and aspartic acid) were added to the N-terminus of the recombinant fusion protein, which are shown in FIG. 13c by underlining. Two IL. The linker sequence separating the 12 subunits is marked with bold letters in 13c.

To express and secrete the mIL-12 fusion protein, eg, in the supernatant of the growth medium, a signal peptide was added to the N-terminus as described above to create a recombinant mIL-12 precursor protein.

The respective amino acid sequences of the recombinant mIL-12 fusion protein precursor are shown in FIG. 13d and SEQ ID NO: 27. The underlined amino acids shown in FIG. 13d mark the secretory signal of the Lactococcus protein Usp45.

1.6.2 Mouse interleukin-18 (mIL-18)
L. The amino acid sequence of the mIL-18 variant that should be expressed by lactis is shown in FIG. 10b and SEQ ID NO: 17. The respective amino acid sequences of the recombinant mIL-18 precursor protein are shown in FIG. 10c and SEQ ID NO: 18. The underlined amino acids shown in FIG. 10c refer to the secretory signal of the Lactococcus protein Usp45.

1.6.3 Mouse interferon alpha-2 (mIFNa2)
The mature form of mIFNa2 contains 167 amino acids, which correspond to amino acids 24-190 of the amino acid sequence of the mouse interferon alpha-2 precursor available in NCBI reference sequence: NP_034633.2.

The amino acid sequence of mature mouse IFNa2 is shown in FIG. 14a and SEQ ID NO: 28.

L. The miFNa2 variant, which should be expressed by lactis, contains an additional alanine residue at the N-terminus of the mature protein to optimize cleavage of the signal peptide. The corresponding amino acid sequences of the recombinant mouse IFNa2 variant to be expressed are shown in FIG. 14b and SEQ ID NO: 29. The underlined amino acids shown in FIG. 14b refer to the additional alanine residue at the N-terminus of the amino acid sequence.

To express and secrete miIFNa2, eg, in the supernatant of growth medium, a signal peptide was added to the N-terminus as described above to produce a recombinant mIFNa2 precursor protein. The respective amino acid sequences of the synthetic miFNa2 precursor protein are shown in FIG. 14c and SEQ ID NO: 30. The underlined amino acids shown in FIG. 14c refer to the secretory signal of the Lactococcus protein Usp45.

1.6.4 Creation of expression construct Amino acid sequence of recombinant mIL-12 protein precursor shown in FIG. 13d and SEQ ID NO: 27, of recombinant mIL-18 precursor protein shown in FIG. 10c and SEQ ID NO: 18. The amino acid sequence, as well as the amino acid sequence of the recombinant miFNa2 precursor protein shown in FIG. 14c and SEQ ID NO: 30, was translated into the corresponding nucleic acid sequence, whereby the codon usage frequency was L. It was adapted to the general codon usage frequency of Lactis.

For the design of the operon, each of the three genes encoding each precursor protein was provided with its own ribosome binding site for translation initiation and its own signal peptide for protein secretion. , As outlined above, L. The same signal sequence (ssUsp45) encoded by different codons based on the codon decompression and codon frequency of lactis was used.

The ribosome binding site of the gadC gene was used for the first gene of the operon (mIL-12). The second gene (mIL-18) has a ribosome binding site for the ATP synthase subunit gamma (atpG) gene, and the third gene (mIFNa2) has a ribosome binding site for the galactoside O-acetyltransferase (lacA) gene. Was used.

The nucleic acid sequence of the synthetic mTEA construct is shown in FIG. 15 and SEQ ID NO: 31.

The completed gene synthesis product is E.I. It was obtained from BaseClear as a fragment cloned on the coli vector pUC57.

The pUC57 plasmid with the gene synthesis product was digested with restriction enzymes SphI and BglII. The SphI and BglII fragments containing the respective gene synthesis products were isolated by phenol extraction and ethanol precipitation and ligated into the SphI and BglII cut plasmid pAUC1010, resulting in the production of an expression plasmid called pC-mTEA. ..

The nucleic acid sequence of the expression plasmid, called pC-mTEA, is shown in FIG. 16b and SEQ ID NO: 32. A schematic diagram of the expression plasmid pC-mTEA is illustrated in FIG. 16a.

1.7. Creation of expression plasmids for expression of mGM-CSF, mIL-12, and mIL-18 from a single operon Mouse granulocyte-macrophage colony stimulating factor (mGM-CSF), mouse interleukin-12 (mIL-12) ), And mouse interleukin-18 (mIL-18), L. The following nucleic acid sequences were generated for expression by Lactis NZ1330.

1.7.1 Mouse Granulocyte-Macrophage Colony Stimulating Factor (mGM-CSF)
L. The mGM-CSF variant that should be expressed by lactis is shown in FIG. 11b and SEQ ID NO: 20. The underlined amino acids shown in FIG. 11b refer to the additional alanine residue at the N-terminus of the amino acid sequence as described in Section 1.5.2 above. The respective amino acid sequences of the synthetic mGM-CSF precursor protein are shown in FIG. 11c and SEQ ID NO: 21. The underlined amino acids shown in FIG. 11c refer to the secretory signal of the Lactococcus protein Usp45.

1.7.2 Mouse interleukin-12 (mIL-12)
L. The mIL-12 variants that should be expressed by lactis are the mature interleukin-12 subunit beta, which forms the N-terminus of the fusion protein, and the mature interleukin-12 subunit alpha, which forms the C-terminus of the fusion protein. Designed as a fusion protein with, both were separated by a peptide linker as described in Section 1.6.1 above. The respective amino acid sequences of the recombinant mIL-12 fusion protein precursor are shown in FIG. 13d and SEQ ID NO: 27. The underlined amino acids shown in FIG. 13d mark the secretory signal of the Lactococcus protein Usp45.

1.7.3 Mouse interleukin-18 (mIL-18)
L. The amino acid sequence of the mIL-18 variant that should be expressed by lactis is shown in FIG. 10b and SEQ ID NO: 17. The respective amino acid sequences of the recombinant mIL-18 precursor protein are shown in FIG. 10c and SEQ ID NO: 18. The underlined amino acids shown in FIG. 10c refer to the secretory signal of the Lactococcus protein Usp45.

1.7.4 Creation of expression construct The amino acid sequence of the recombinant mGM-CSF precursor protein shown in FIG. 11c and SEQ ID NO: 21 and the recombinant mIL-12 fusion protein precursor shown in FIG. 13d and SEQ ID NO: 27. The amino acid sequence of the body is translated into the corresponding nucleic acid sequence, whereby the frequency of codon usage is L. It was adapted to the general codon usage frequency of Lactis.

For the design of the operon, each of the two genes encoding each precursor protein was provided with its own ribosome binding site for translation initiation and its own signal peptide for protein secretion. , As outlined above, L. The same signal sequence (ssUsp45) encoded by different codons based on the codon decompression and codon frequency of lactis was used.

The ribosome binding site of the gadC gene was used as the first gene of the operon (mGM-CSF). The second gene (mIL-12) has a ribosome binding site for the ATP synthase subunit gamma (atpG) gene, and the third gene (mIL-18) has a galactoside O-acetyltransferase (lacA) gene ribosome. The binding site was used.

The nucleic acid sequence of the synthetic mGTE construct is shown in FIG. 16a and SEQ ID NO: 33.

The completed gene synthesis product is E.I. It was obtained from BaseClear as a fragment cloned on the coli vector pUC57.

The pUC57 plasmids with the respective gene synthesis products were digested with the restriction enzymes SphI and BglII from New England Biolabs (Ipswich, MA, US), respectively. The SphI and BglII fragments containing the respective gene synthesis products were isolated by phenol extraction and ethanol precipitation and ligated into the SphI and BglII cut plasmid pAUC1010, resulting in the production of an expression plasmid called pC-mGTE. ..

The nucleic acid sequence of the expression plasmid, called pC-mGTE, is shown in FIG. 16c and SEQ ID NO: 34. A schematic diagram of the expression plasmid pC-mGTE is illustrated in FIG. 16b.

Example 2 Production of recombinant bacteria The expression plasmid obtained in Example 2 was obtained by means of electroporation as outlined above. It was transformed into a lactis strain. Each L. used in the following example. Lactis strains are summarized in Table 1 below.

For further testing, each recombinant bacterial stock with a cell density of approximately 1 × 10 ¹¹ colony forming units (CFU) / ml is 20 wt after growth on IM1 or CDM3 medium with lactose and Na beta-glycerophosphate. Prepared by the addition of% final concentration of glycerol. The stock was stored at -80 ° C.

The composition of the CDM3 medium was Zhang, G. et al. and Block, DE ( "Using highly efficient nonlinear experimental design methods for optimization of Lactococcus lactis fermentation in chemically defined media", Biotechnol.Prog.2009,25 (6): pages 1587 to 1597, doi: 10.1002 / btpr.277 ) Is based on the ZMB3 medium described in.

Example 3 In an in vitro Pgad promoter activity determination induction study, IM1 medium was indicated in Table 1 for each L. It was used to grow the Lactis NZ1330 strain. IM1 medium was replenished as described below in the next experiment:
2 wt% β-glycerophosphate (Sigma, Catalog no. 50020-500G)
1.5 wt% soybean peptone (BD, catalog no. 211906)
1 wt% yeast extract (BD, catalog no. 221750)
1 mM י ₄ . 7H ₂ O (Merck, catalog number. 10588860500)
0.1 mM י ₄ . 7H ₂ O (Sigma, catalog no. M-7634)
3 wt% glucose (Natural Spices B.V., catalog no. ES212)

In pH controlled fermentation, 3.5 M NH ₃ OH was used for base addition.

Induction protocol Frozen stock from cell banks was used to inoculate 50 ml of IM1 medium supplemented with 3% glucose and then incubated at 30 ° C. for 16 hours (preculture). These overnight cultures were used for inoculation (1 vol% inoculation) into a 100 ml fermenter charged with IM1 medium with 3% glucose, pH 6.5, 30 ° C. The cultures were mixed at a continuous speed of 200 rpm using magnetic stirrer.

Expression of each gene was induced by 100 mM NaCl at OD ₆₀₀ = 0.5. Control cells were grown on additional NaCl-free medium and therefore expression of each gene was not induced.

L. The effect of chloride-induced expression of the heterologous gene on the viability of Lactis NZ1330 is the NaCl-induced L. It was assessed by comparing the growth curves of Lactis NZ1330 and uninduced controls.

Cultures were sampled at 0.5, 1, 2, 3, 4, 5, and 6 hours. These samples were used for OD ₆₀₀ measurements and mCherry activity determination. Bacterial growth was monitored by measuring 600 nm optical density (OD600) of either induced bacterial cells or underived controls. A comparison of each growth curve is shown in FIG. 19a.

mCherry fluorescence activity was measured using a Synergy HT fluorescence spectrophotometer (Biotek, Winooski, Vermont, United States). Excitation was at 530 nm, emission was measured at 590 nm and a gain of 120 was applied.

The results are shown in FIG. 19b. As can be seen in FIG. 19b, the induction of mCherry gene expression by the addition of 100 mM NaCl resulted in a significant increase in fluorescence, which was L. Corresponds to the increased amount of mCherry protein produced by chloride-induced expression of the heterologous gene in Lactis NZ1330. The increased amount of mCherry protein expression did not significantly affect the viability of each bacterium after induction of mCherry gene expression, because, as can be seen in FIG. 19a, the induced bacterial growth curve This is because they did not show a significantly reduced growth rate compared to uninduced bacteria.

Chloride-induced expression of FGF-2, IL-4, and CSF-1 from a recombinant strain called AUP-1602-C is +4 by Western blotting using the protocol indicated above. It was determined from the TCA precipitate of 1 ml supernatant obtained by centrifugation of each culture at 14000 g for 10 min at ° C.

Purified preparations of commercially available recombinant human CSF-1 protein (Abcam-PLC, 5 ng), recombinant human FGF-2 protein (R & D Systems, 7.5 ng), and recombinant human IL-4 protein (Sigma Aldrich, 5 ng). Was used as a positive control standard. Each primary antibody used is indicated above. As the secondary antibody, an HRP-linked antibody (catalog Nr. # 7074) of anti-rabbit IgG from New England Biolabs was used.

Each Western blot is shown in FIG. 20a. As can be seen in FIG. 20a, all three recombinant proteins were expressed by AUP-1602-C and released into the supernatant.

Chloride-induced expression of GM-CSF from a recombinant strain called AUP5563-C by Western blotting using the protocol indicated above, each culture at + 4 ° C. for 10 min at 14000 g. It was determined from the TCA precipitate of 1 ml supernatant obtained by centrifugation in. A purified preparation of a commercially available recombinant mouse GM-CSF protein (Sigma-Aldrich) was used as a positive control standard.

Each Western blot is shown in FIG. 20b. As can be seen in FIG. 20b, GM-CSF is expressed by AUP5563-C and released into the supernatant.

Chloride-induced expression of IL-18 from a recombinant strain called AUP5563-C by Western blotting using the protocol indicated above, each culture at 14000 g for 10 min at + 4 ° C. It was determined from the TCA precipitate of 1 ml supernatant obtained by centrifugation in. A purified preparation of a commercially available recombinant mouse IL-18 protein (R & D Systems) was used as a positive control standard.

Each Western blot is shown in Figure 20c. As can be seen in FIG. 20c, IL-18 is expressed by AUP5563-C and released into the supernatant.

Chloride-induced expression of IL-12 from recombinant strains called AUP5551m-C and AUP5563-C by Western blotting using the protocol indicated above at + 4 ° C. at 14000 g for 10 min. It was determined from the TCA precipitate of 1 ml supernatant obtained by centrifugation of each culture. A purified preparation of a commercially available recombinant mouse IL-12 protein (R & D Systems) was used as a positive control standard.

Each Western blot is shown in FIG. 20d. As can be seen in FIG. 20d, IL-12 is expressed by AUP5551m-C and AUP5563-C and released into the supernatant.

Example 4 Determining the activity of the in vivo Pgad promoter The activity of the in vivo Pgad promoter system utilizes chloride-induced mCherry constructs and measures the fluorescence signal of the in situ after intratumoral (it) injection. Was evaluated by.

Mouse CT26 is an N-nitroso-N-methylurethane (NNMU) -induced undifferentiated colon cancer cell line from mice. It was cloned into CT26. A cell line called WT (ATCC® CRL-2638 ™) was created, which is commercially available from LGC Standards GmbH.

When inoculated into BALB / c mice, CT26. WT cells can induce lethal tumors.

Wang, M.M. et al. According to (1995), BALB / ^c mice subcutaneously inoculated with CT26 cells developed lethal tumors at 80% frequency with ¹⁰³ cells and 100% with 104 cells. Lung metastases occurred when mice were intravenously inoculated with ¹⁰⁴ cells. BALB / cJ mice (genotype: A / A Typ1b / Typ1b Typ / Typ, Stock Code000651) commercially obtained from The Jackson Laboratory (Bar Harbor, ME, USA) were used in this study.

BALB / c mice can be CT26. Inoculated with WT cells.

1 × ^10-5 tumor cells in 0.1 ml PBS were subcutaneously injected into the left flank of the mouse. Treatment was initiated when the tumors reached an average volume of ~ 100-200 mm ³ , after which mice were assigned to those treatment groups with a uniform tumor average volume between the groups.

L. cerevisiae containing the pC-mCherry construct. After a single intratumoral injection of Lactis NZ1330, tumor-bearing mice were imaged using IVIS Spectrum CT (excitation filter: 535-570 nm / luminescence filter: 580-680 nm). The animals are L. Lactis NZ1330 (pC-mCherry) bacterium, or control bacterium, ie L. Lactis NZ1330, i. t. Imaging was performed 48 hours after injection. The length of image acquisition and binning (sensitivity) are dependent on the intensity of the lesions present and are captured and processed using Living Image 43.1 software (PerkinElmer, Inc., Waltham, MS, US). rice field.

The results are shown in FIG. 21, which are described in L. et al. Lactis NZ1330 (pC-mCherry) and control bacteria i. t. Fluorescence imaging 48 hours after injection is shown. The black circle indicates the location of the tumor. Higher mCherry activity is indicated by the black areas.

As can be seen in FIG. 21, a single 50 μl intratumoral injection of 2 × 10 ¹⁰ CFU / ml recombinant bacteria obtained in Example 3 and expressing the mCherry protein under the control of the PgadC promoter system is each in the tumor environment. Provided sufficient localization and survival of the bacteria.

The results are that the recombinant bacteria of the invention express and secrete at least one therapeutic protein, thereby affecting the cells of the innate and / or adaptive immune system of the tumor environment to achieve an antitumor response. It clearly shows that it can survive long enough in the environment.

Moreover, the chloride concentration within the tumor environment is high enough to provide expression of each at least one therapeutic protein that should be expressed by the recombinant bacteria of the invention.

Example 5: Wound Closure Experiment Diabetic patients tend to heal impaired wounds, with ulcerization of the foot predominant. This delay in wound healing also applies to diabetic animals, including spontaneously diabetic (db / db) mice commercially obtained from The Jackson Laboratory (Bar Harbor, ME, US).

Sixty diabetic mice, all male and approximately 10 weeks old (strain name BKS.Cg-Dock7m +/+ Leprdb / J-Stock Code00642) were used in the first study.

The consequences of the application of bacteria expressing the combination of human FGF-2, CSF1, and IL-4 to excised wounds of all layers of db / db diabetic mice under the control of the PgadC promoter, respectively, are pharmacodynamic (PK). ) / Pharmacodynamic (PD) combination efficacy study.

In it, a recombinant bacterium called AUP16-C obtained in Example 3 expressing FGF-2, IL-4, and CSF-1 by a single bacterial cell was evaluated and compared to vehicle treatment. .. The composition of each vehicle used in the application is summarized below:
Vehicle 1: 0.9% saline 5% dextrose Vehicle 2: 5% dextrose + 0.9% saline + 200 mM Na acetate (pH 6.5)
Vehicle 3: 5% dextrose + 2.5% saline Vehicle 4: 5% dextrose + 2.5% saline + 200 mM Na acetate (pH 6.5)
Vehicle 5: 10% dextrose + 0.9% saline Vehicle 6: 10% dextrose + 0.9% saline + 200 mM Na acetate (pH 6.5)

Animals were randomized to one of the seven treatment regimens according to Table 2.

Recombinant bacteria expressing FGF-2, IL-4, and CSF-1 by a single bacterial cell were applied to the wound on the day of injury (day 0) and their survival 6 hours after application. It was investigated.

Wound fluid samples are taken from the wound after 6 hours, and 1, 2, and 7 days to determine the production of FGF-2, CSF-1, and / or IL-4 by the bacteria applied to the wound. rice field. To aid in PK / PD analysis, systemic blood and major organs were harvested after 6 and 24 hours and 7 days. Wounds that received vehicle 1 alone were used as controls for the PK / PD components of this study.

To investigate the impact of these recombinant bacteria on the process of wound healing, the wounds were wounded by bacteria expressing human FGF-2, CSF-1, and IL-4 on the day they were injured (day 0), and every day thereafter. It was applied to the wound until the 6th day later. Healing of wounds exposed to these recombinant bacteria was compared to that of similar wounds exposed only to new vehicle 1.

Wound healing was studied at both the macroscopic and histological levels. Wound healing was studied at the macroscopic level in terms of initiation of neodermal repair response, wound contraction, and wound closure.

After wounding, wound closure and wound contraction and wound re-epithelialization of its components were determined from digital photographs taken on the 0th, 4th, and 7th days after the wound. Histological assessments of granulation tissue formation (depth) and wound width (head-to-tail contraction) were made on routine (H & E) stained sections. These histological assessments were made on tissues harvested on the 7th day after wound.

The occurrence of harmful effects was monitored and fully recorded.

Full-thickness experimental wound generation and application of treatment All mice were anesthetized with isoflurane and air; their dorsal flank skin was depilated and cleaned. A single standardized full-thickness wound (10 mm x 10 mm) was created on the left flank approximately 10 mm from the spine. Each wound was then imaged with an identification plate and a graduated ruler.

All wounds were then covered with a clear film dressing Tegaderm® Film (3M Deutschland GmbH, Neuss, DE). The animals were then allowed to recover in a warm environment (34 ° C). Animals were later retained and dosed with one of the procedures listed in Table 2, each applied by injection through a Tegaderm® film using a 27 gauge needle.

The procedure was similarly reapplied to the wounds daily until the 6th day after the wound, therefore each wound received 7 applications. All wounds are closely monitored for excessive build-up and excessive wound site hydration of the applied drug; excess products / fluids are on the 4th, 6th, and 8th days after the wound, and 4th. And removed by inspiration prior to reapplication on the 6th and prior to dressing removal on the 8th day.

On the 4th, 8th, 12th, and 16th days after the wound, all animals were re-anesthetized, their film dressings, and any free debris were gently removed, and the wounds were gauze soaked in sterile saline. Was cleaned using. The wound was then imaged and recoated with Bioclusive® film dressing (as above), allowing the animal to heal in a warm environment (34 ° C.).

Immediately after wounding, and on the 4th, 8th, and 12th days after the wound, all wounds were digitally imaged with a graduated / identification plate after film dressing removal and wound cleaning.

All animals were slaughtered on the 20th day after wound imaging. The culling was accomplished by a method that complied with the UK Ministry of Home Affairs "Schedule 1".

Image Analysis of Wound Closure Each wound, along with an identification / graduated plate, was digitally imaged immediately after the wound and on the 4th, 8th, 12th, 16th, and 20th days after the wound, and the open wound area was measured and the first. Expressed as% wound area relative to day 0.

Image Pro Plus image analysis software (version 4.1.0.0, Media Cybernetics, Inc., Rockville, MD, US) was used to calculate wound closure from scaled wound images taken at each evaluation time point.

The results of each treatment group are summarized in Table 3 and FIG.

For a given wound at a given time point, wound closure was expressed as the remaining percentage of the wound area relative to the initial wound area immediately after the injury (ie, day 0). Table 3 shows the average percentage of the remaining wound area in all treatment groups.

As can be seen from the data submitted in Table 3, all groups that received AUP1602-C bacteria were control groups (5% dextrose in 0.9% physiological saline) at all time points, regardless of the delivery vehicle. ) Was found to close significantly more rapidly.

Wound contraction Contraction is the central movement of the wound margin due to consolidation of granulation tissue within the "body" of the wound.

The "consolidation" force that drives this process is thought to be possessed by cells of the fibroblast lineage. In this study,% shrinkage is:
% Shrinkage = Area defined by the boundary between normal dermis and "repair new dermis" x 100 / original wound area (Day 0)
Calculated as.

Mean percentage wound contraction data for all treatment groups are shown in Table 4 and FIG.

As can be seen from Table 4, all groups receiving AUP1602-C bacteria were significantly faster than the control group (5% dextrose in 0.9% physiological saline) at all time points, regardless of the delivery vehicle. It was found to shrink to (p ≦ 0.007).

Wounds in non-diabetic mice are closed primarily by contraction, whereas those in diabetic mice have a significantly reduced ability to contract, probably due to poor granulation tissue formation. As a result, diabetic wounds tend to be closed by re-epithelialization to a greater extent than non-diabetic animals. The forces driving the process of contraction are thought to be derived from the activity of fibroblasts resident in the neodermal compartment of the skin wound.

Observations of enhanced contractions after application of AUP1602-C clearly suggest an improvement in granulation tissue function; in turn, an increase in the amount of granulation tissue formed, the speed at which it is formed. And / or the increased contractility of the tissue.

Initiation of wound healing (new dermal tissue formation)
Wounds were visually assessed daily until day 8 and then every other day to establish a "healing" status. Each wound was scored as to whether it displayed "new dermal tissue-forming activity", i.e., whether the wound initiated the dermal healing process. Each wound was assessed by two independent observers, and the percentage of wounds displaying "new dermal tissue formation activity" was compared between treatment groups at each assessment time point.

The number of responsive wounds for each treatment group for each day is shown in Table 5.

The percentage of responsive wounds in each treatment group on day 1 is shown in FIG. In the majority of wounds that received AUP1602-C, a healing response was apparent after the first day of dosing on the first day after the wound, regardless of the delivery vehicle used.

On the first day after the wound, treatment groups AUP1602-C (Veh2), AUP1602-C (Veh3), and AUP1602-C (Veh6) 100% of the wound, AUP1602-C (Veh4) 88%, AUP1602-C. It was found that 80% of (Veh1) and 75% of AUP1602-C (Veh5) initiated a healing response.

No significant difference was detected between these treatment groups (Fisher's exact test).

It was found that 100% of the wounds that received AUP1602-C demonstrated a healing response by the second day after the wound.

None of the wounds that received vehicle 1 alone (5% dextrose in 0.9% physiological saline) demonstrated a healing response during the 20-day study period.

It was found that in all AUP1602-C treatment groups compared to the vehicle control group, a significantly greater proportion of wounds responded at all time points evaluated (p ≤ 0.001, Fisher's exact test). Accurate test).

A significantly higher proportion of wounds exposed to AUP1602-C bacteria, regardless of formulation, compared to those observed in the vehicle 1 control group (5% dextrose alone in 0.9% physiological saline). "New dermal tissue formation" was initiated, i.e., 100% of wounds that received AUP bacteria responded; wounds that received vehicle 1 alone did not appear to respond.

For all compound vehicles, AUP1602-C treatment initiated a response in the wound bed very early (within 1-2 days) after application.

Angiogenesis Response Wound images on days 4 and 8 were visually evaluated to quantify the level of angiogenesis. Mean scores for angiogenic responses on days 4 and 8 of all groups are shown in Table 6, and scores from two independent observers were averaged.

All treatments involving AUP1602-C were scored significantly higher for angiogenesis than vehicle 1 alone (p≤0.002) when observed on days 4 and 8.

Wounds infected with AUP1602-C bacteria were evaluated at both day 4 and day 8 compared to vehicle 1 control group (5% dextrose alone in 0.9% physiological saline), regardless of vehicle. Demonstrated significantly increased angiogenesis.

All bacterial treatment regimens examined in this experiment were used in diabetic db / db mice, one of the widely accepted and best-validated animal models of delayed wound healing in humans, regardless of vehicle. It was found to promote wound repair.

Resected wounds in db / db mice show a statistically significant delay in wound closure, reduced granulation tissue formation, reduced wound bed vascular distribution, and markedly diminished proliferation, such as Michaels et al. (2007). be.

Experimental results clearly show that the application of the recombinant bacteria of the present invention provides a significant improvement in the treatment of human wounds, especially chronic wounds.

Application of the recombinant bacteria of the present invention induces granulation tissue formation, increased wound bed vascular distribution, and proliferation, thus leading to improved and accelerated wound closure even in models showing severe wound healing damage. ..

Therefore, these results are inflammatory, preferably chronic inflammatory skin disorders such as frost, eczema, psoriasis, dermatitis, ulcers, wounds, systemic erythematosus, neurodermatitis, and combinations thereof, preferably. It has been shown that dermatitis, ulcers, wounds, and combinations thereof, and more preferably ulcers, benefit from the application of the recombinant bacteria of the invention.

Chronic wounds such as, for example, chronic venous ulcers, chronic arterial ulcers, chronic diabetic ulcers, and chronic pressure ulcers can be treated by application of the recombinant bacteria of the invention, because the wounds observed in each chronic wound. This is because healing damage is preferably overcome after application of the recombinant bacteria of the invention expressing human FGF-2, human IL-4, and human CSF-1.

Example 6: Detection of human FGF-2, human IL-4, and human CSF-1 in wound fluid.
As in Example 5, all male and approximately 10 week old diabetic mice (strain name BKS.Cg-Dock7m + // + Leprdb / J-Stock Code00642) were used in subsequent studies.

Detection of each recombinant protein was investigated in this study after application of bacteria expressing the combination of human FGF2, CSF1, and IL4 to excised wounds of all layers of db / db diabetic mice, respectively, under the control of the PgadC promoter. Was done. In it, a recombinant bacterium called AUP16-C obtained in Example 3 expressing FGF-2, IL-4, and CSF-1 by a single bacterial cell was evaluated for vehicle composition. The composition of each vehicle used in the application is summarized below:
Vehicle 1: 0.9% 5% dextrose in physiological saline Vehicle 4: 2.5% 5% dextrose in physiological saline
+200 mM Na acetate (pH 6.5)
Vehicle 7: 5% dextrose in 2.5% physiological saline
+200 mM Na acetate
+ 50 mM L-glutamic acid (pH 6.0)

Wound fluid samples were prepared from Sigma-Aldrich by a commercially available 5 kDa cutoff concentrator (Vivaspin® sample concentrator) using 0.5% SDS according to the manufacturer's protocol.

Samples were separated by SDS PAGE gel and above I.I. It was blotted on nitrocellulose as described in Section 8.

Detection of recombinant protein was performed by using the following antibody.

Purified mouse anti-human FGF-2 monoclonal antibody (BD Transition laboratories, Heidelberg, DE) was used for the detection of human FGF-2 at a dilution of 1: 250. The secondary antibody is Bioke B. V. (Leiden, NL) was a 1: 10,000 diluted horseradish peroxidase (HRP) -linked antibody, anti-mouse IgG.

For the detection of human IL-4, monoclonal mouse anti-human IL-4IgG1 (clone # 3007) from R & D systems was used at a 1: 500 dilution. The secondary antibody is Bioke B. V. (Leiden, NL) was a 1: 10,000 diluted HRP-linked antibody anti-mouse IgG.

Rabbit polyclonal antibody (clone ab9693) against human MCSF from Abcam PLC was used for the detection of human CSF-1 at a dilution of 1: 2,000. The secondary antibody is Bioke B. V. It was an HRP-linked anti-rabbit IgG antibody at a dilution of 1: 2,000 obtained from (Leiden, NL).

The amount of each recombinant protein in the wound fluid was determined by comparison with each reference protein.

The human FGF-2 reference protein was obtained from Sigma Aldrich and was lysed in 5 mM Tris pH 7.5 to give a final concentration of 100 μg / μl. For reference, a total amount of 10 mg, 3.3 ng, 1.1 ng, 0.37 ng, and 0.12 ng was applied to different lanes of Western blot. The results are shown in FIGS. 25a and 25b.

The human IL-4 reference protein was obtained from R & D systems (Minneapolis, MN, US). Human IL-4 was dissolved in PBS (5.8 mM Na ₂ HPO ₄ , 4.2 mM NaH ₂ PO ₄ , 145 mM NaCl) to give a final concentration of 100 μg / mL. For reference, a total amount of 10 mg, 3.3 ng, 1.1 ng, 0.37 ng, and 0.12 ng was applied to different lanes of Western blot. The results are shown in FIGS. 26a and 26b.

The human CSF-1 reference protein was obtained from Abcam PLC (Cambridge, GB). Human CSF-1 was dissolved in sterile water to give a final concentration of 100 μg / mL. For reference, a total amount of 10 mg, 3.3 ng, 1.1 ng, 0.37 ng, and 0.12 ng was applied to different lanes of Western blot. The results are shown in FIGS. 27a and 27b.

Recombinant bacteria using the PgadC system adequately expressed human FGF-2, human IL-4, and human CSF-1 in vivo, and all three target proteins could be detected in wound fluid.

AUP1602-C with the PgadC promoter system, under in vivo conditions, without the requirement of additional administration of external inducers and / or excipients such as β-galactosidase or nisin after topical application of each recombinant bacterium. , Produced a sufficient amount of each target protein.

Example 7 Inhibition of cancer cell growth in vivo The premise of successful immunotherapy is the presence of a fully functional immune system. In that, the immunogenicity of the tumor is regarded as a powerful feature that predicts the response to immunotherapy.

Among the different mouse models developed to assess the properties of anticancer agents and / or treatments, the syngenic model is a mouse that carries a genetically identical murine lineage tumor. These models provide an intact immune and microenvironmental system for testing immunotherapeutic agents and / or antiangiogenic agents and treatment regimens.

The background mouse strains used to generate each murine model are that tumors established in BALB / c mice are generally more immunogenic than tumors established in, for example, C57BL / 6 mice, and tumors. It has been shown to affect immunogenicity.

Furthermore, Lechner, M. et al. G. et al. The study of the murine solid tumor model performed by (2013) shows that among the different cell lines, CT26, RENCA, and 4T1 tumors are the most for immunotherapy regimens that include significantly reduced tumor growth and increased survival. Demonstrated that it shows a large positive response.

Therefore, CT26 cells were selected as the cell line to be used in BALB / C mice to initially assess the efficacy of administration of AUP5563-C4 in vivo.

Efficacy studies with AUP5563-C4 expressing IL-12, IL-18, and GM-CSF were performed to evaluate the effect of intratumoral administration of the bacterial preparation AUP5563-C4 in the treatment of subcutaneous murine CT-26 cancer models. did.

As described in Example 4 above, CT26. WT cells were used to induce lethal tumors in BALB / c mice, but ¹ × 105 CT26 in 0.1 ml PBS. WT cells were injected subcutaneously into the left flank of the mouse with a 27-gauge needle.

All comments regarding body weight, dosing, and clinical status were captured in real time using the study management software StudyDirector (StudyLog Systems, Inc., San Francisco, CA, US).

Tumors are measured three times weekly during the procedure and the apparent tumor volume is formula V = 0.5 x (length x width ² ).
In the formula, V is the tumor volume, L is the tumor length, and W is the tumor width, eg, Tomayko, M. et al. and Reynolds, C.I. As described in (1989), tumors were measured in two dimensions using an electronic caliper.

Mice were randomly assigned to the treatment group. Treatment was initiated when the tumor reached an average volume of 50-100 mm ³ . Mice were assigned to those treatment groups 1 to 3 with a uniform average tumor volume between the groups. The duration of treatment was up to 2 weeks. The treatment groups are summarized in Table 7.

Efficacy was assessed after intratumoral (it) injection of AUP5563-C4 (1 × ¹⁰⁸ CFU) three times weekly over a two-week period.

The vehicle-treated group was used as a negative control, and the anti-CTLA-4 treated mice were used as a positive control. Monochromic anti-mouse CTLA-4 antibody clone 9D9 was obtained from Bio X Cell (West Lebanon, NH, US), a mouse cytotoxic T lymphocyte-related protein 4 (mouse cytotoxic T lymphocyte-related protein 4 that is a protein receptor that down-regulates the immune system. Orient to CTLA-4).

The results are summarized in FIGS. 28a-28c, which are (a) saline (negative control), (b) AUP5563-C4, and (c) mouse anti-CTLA4 (positive control) tumors, respectively. It shows the evaluation of tumor growth after administration (it).

As can be seen in FIGS. 28a and 28b, i.S. of AUP5563-C4 (1 × 10 ⁸ CFU) three times weekly over a two-week period. t. Injection significantly reduced tumor growth compared to vehicle-treated control mice.

Administration of AUP5563-C4 (1 × ¹⁰⁸ CFU) three times weekly over a two-week period resulted in a reduction in tumor growth comparable to treatment with the anti-m-CTLA-4 antibody used as a positive control.

The results are i.I. of AUP5563-C4. t. It demonstrates that administration had similar efficacy to the immune checkpoint inhibitor anti-CTLA4.

Tumors that received intratumoral injections of saline (negative control) were growing well.

Example 8 Treatment of Intraperitoneal Tumors in Mice To evaluate the feasibility and efficacy of recombinant bacteria of the invention expressing at least one therapeutic protein under the control of the Pgad promoter after intraperitoneal administration, B16-F1 Cells were intraperitoneally inoculated into C57BL / 6 mice and survival as a primary endpoint was measured.

B16 melanoma cells were selected for tumor inoculation, because Fu Q. et al. This is because C57BL / 6 mice are syngenic hosts of B16-F1 cells with high tumor uptake rates, as described by (2016).

Moreover, this model is described by Lesinski, G. et al. B. It has been shown to respond to immunotherapeutic treatment regimens as described by et al (2003).

Inoculation of mouse B16-F1 cells into C57BL / 6 mice leads to the development of malignant tumors, which can kill the mice before day 20 after transplantation without any treatment.

Mouse B16-F1 (KCLB no80007) was obtained from the Korean Cell Line Bank. Cells were seeded in 96-well microplates and incubated for 24 hours under the following conditions:
-Culture conditions: 37 ° C.
Culture medium: Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies Corporation, Carlsbad, CA, US) supplemented with 50 U / ml of penicillin, 50 μg / ml of streptomycin, and 10% by volume of fetal bovine serum.

B16-F1 cells are a well-known model for the study of metastasis and solid tumorigenesis.

Orient Bio Inc. A total of 66 C57BL / 6N mice commercially obtained from (Gyeonggi-do, KR) were used in this study.

1 × 10 ⁵ B16F1 cells were inoculated into each individual C57BL / 6 mouse by intraperitoneal (ip) injection (day 0). Treatment with either vehicle (5% dextrose in 0.9% physiological saline) or AUP2059 / AUP5551-C (1: 1) is given intraperitoneally three times weekly starting on day 0 for up to 5 weeks. (300 μl per injection) (up to 15 doses during days 0 and 35).

Animals were randomized to one of the six treatment regimens according to Table 8.

Treatment was performed by administration of a combination of the drug products AUP2059 (mIL18 / mGM-CSF) and AUP5551-C (mIL12 / mIL18 / mIFNa2b) obtained in Example 3, respectively.

Recombinant bacteria were mixed in a 1: 1 ratio based on CFU / mL in total amounts of 2 × 10 ⁵ , 2 × 10 ⁶ , 2 × 10 ⁷ or 2 × 10 ⁸ CFU per 300 μl dose.

Treatment was performed by Dai Han Pharm. CO. 1: 1 combination of AUP2059 (mIL18 / mGM-CSF) and AUP5551-C2 (mIL12 / mIL18 / miFNa2b) in commercially available 5% dextrose in a 0.9% aqueous saline solution obtained from (Dai Han, KR). I. It was administered by p-injection.

All dosing was started on day 0 after inoculation of B16F1 cells.

The average survival time compared to vehicle treatment is summarized in FIG.

As can be seen in FIG. 29, treatment with the combination of AUP2059 (mIL18 / mGM-CSF) and AUP5551-C2 (mIL12 / mIL18 / mIFNa2b) resulted in a significant increase in survival time compared to vehicle-treated mice. To reach.

Syngenic cancer cell transplantation induced rapid mortality in vehicle-treated controls, with all animals dying by day 19.

In comparison, treatment with the combination of AUP5551-C2 and AUP2059 increased the survival of mice in a dosing-dependent manner. Survival was improved by more than 70% with an average of 28.3 and 28 days, respectively, and median survival. These results indicate that administration of the recombinant bacteria of the invention, eg, a combination of AUP5551-C2 and AUP2059, is highly effective in delaying tumor growth in syngenic mice with intraperitoneal (IP) tumors. It is clearly shown.

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Claims (31)

It ’s a recombinant bacterium,
a) At least one nucleic acid sequence that is functionally coupled to a chloride-inducible promoter of a prokaryotic system and encodes at least one heterologous factor, the heterologous factor being independently a heterologous polypeptide or complex thereof. ,
b) At least one prokaryotic regulator gene that controls the activity of the chloride-inducible promoter,
Including
The heterologous polypeptide comprises at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof.
Recombinant bacteria. Recombinant nucleic acid
a) At least one nucleic acid sequence that is functionally coupled to a chloride-inducible promoter of a prokaryotic system and encodes at least one heterologous factor, the heterologous factor being independently a heterologous polypeptide or complex thereof. ,
b) At least one prokaryotic regulator gene that controls the activity of the chloride-inducible promoter,
Including
The heterologous polypeptide comprises a eukaryotic polypeptide, at least one fragment thereof, or a combination thereof.
Recombinant nucleic acid. At least one nucleic acid sequence that is functionally coupled to a chloride-inducible promoter of a prokaryotic system and encodes at least one heterologous factor, and / or at least one prokaryote that controls the activity of the chloride-inducible promoter. The recombinant bacterium according to claim 1, wherein the system regulator genes are independently located on the chromosome of the recombinant bacterium and / or at least one plasmid. The recombinant bacterium or claim according to any one of claims 1 or 3, wherein the chloride-inducible promoter and / or at least one regulator gene is independently, preferably from a Gram-positive or Gram-negative bacterial species, respectively. Item 2. The recombinant nucleic acid according to item 2. The recombinant bacterium or claim 2 according to any one of claims 1 or 3 to 4, wherein the chloride-inducible promoter is a taxonomic species of the order Lactobacillus, preferably PgadC from Lactococcus lactis. Alternatively, the recombinant nucleic acid according to any one of 4. The recombinant bacterium according to any one of claims 1 or 3 to 4, wherein the regulator gene encodes a gadR from a taxonomic species of the order Lactobacillus, preferably Lactococcus lactis. 4. The recombinant nucleic acid according to any one of 4. A chloride-inducible promoter and at least one regulator gene are located on a chloride-inducible gene expression cassette, whereas a chloride-inducible promoter is located downstream from at least one regulator gene to induce chloride. The recombinant bacterium according to any one of claims 1 or 3 to 6, wherein the sex gene expression cassette controls transcription of at least one nucleic acid sequence encoding at least one heterologous factor or claims 2 or 4 to 6. The recombinant nucleic acid according to any one of the above items. The recombinant bacterium according to claim 7, wherein the chloride-inducible gene expression cassette contains or consists of the nucleic acid sequence of SEQ ID NO: 2 and controls the transcription of at least one nucleic acid sequence encoding at least one heterologous factor. Or recombinant nucleic acid. The recombinant bacterium according to any one of claims 1, 3, or 7 to 8, or any one of claims 2 or 7 to 8, wherein the at least one heterologous factor has a therapeutic and / or preventive effect in the subject. Recombinant nucleic acid according to the section. Claims 1, 3, or 7 where at least one heterologous factor is a heterologous polypeptide or complex thereof, each comprising at least one eukaryotic polypeptide, at least one fragment thereof, or a combination thereof. The recombinant bacterium according to any one of claims 2 to 9 or the recombinant nucleic acid according to any one of claims 2 or 7 to 9. At least one heterologous factor is a polypeptide hormone, growth factor, cytokine, chemokine, enzyme, neuropeptide, antibody, precursors thereof, fragments thereof from eukaryotic species, preferably mammalian species, more preferably humans. , And the recombinant bacterium according to any one of claims 1, 3, or 7 to 10, or the recombinant according to any one of claims 2 or 7 to 10, selected from the group consisting of a combination thereof. Nucleic acid. The recombinant bacterium according to any one of claims 1 or 3 to 11, further comprising at least one inactivated gene encoding an essential protein required for the survival of the recombinant bacterium. Claim that the gene encoding an essential protein is inactivated by gene deletion, gene mutation, gene silencing mediated by RNA interference (RNAi) of the gene, inhibition of gene translation, or a combination thereof. 12. The recombinant bacterium according to 12. The recombinant nucleic acid according to any one of claims 2 or 4 to 11, further comprising at least one gene encoding an essential protein required for the survival of the recombinant bacterium. At least one gene required for viability consists of alanine racemase (all), thymidylate synthase (thyA), aspartate synthase (asnH), CTP synthase (pyrG), tryptophan synthase (trpBA), and combinations thereof. The recombinant bacterium according to any one of claims 12 to 13 or the recombinant nucleic acid according to claim 14, which is selected from the group. The nucleic acid according to any one of claims 2, 4 to 11, 14, or 15, wherein the recombinant nucleic acid is in the form of a plasmid. The recombinant bacterium according to any one of claims 1, 3 to 13, or 15, comprising at least one recombinant nucleic acid according to any one of claims 2, 4 to 11, 15, or 16. The recombinant bacterium according to any one of claims 1, 3 to 13, 15, or 17, wherein the recombinant bacterium is a non-pathogenic bacterium. The recombinant bacterium according to any one of claims 1, 3 to 13, or 15 to 18, wherein the recombinant bacterium is a recombinant bacterium, preferably a lactic acid bacterium, and more preferably a Lactobacillus or Lactococcus species. The recombinant bacterium according to any one of claims 19, wherein the Lactococcus species is Lactococcus lactis, preferably Lactococcus lactis subspecies Cremoris. The recombinant bacterium according to any one of claims 1, 3 to 13, 15, or 17 to 21, for medical use. The recombinant bacterium according to any one of claims 1, 3 to 13, 15, or 17 to 21, preferably for use in the treatment of chronic inflammatory wounds. The recombinant bacterium according to any one of claims 1, 3 to 13, 15, or 17 to 21, for use in the treatment of tumors, preferably malignant tumors. A pharmaceutical composition comprising the recombinant bacterium according to any one of claims 1, 3 to 13, 15 or 17 to 21, and at least one pharmaceutically acceptable excipient. a) The recombinant bacterium according to any one of claims 1, 3 to 13, 15 or 17 to 21, capable of expressing at least one heterologous factor under the control of a chloride-inducible promoter of a prokaryotic system. ,
b) At least one inducing factor, including chloride ion,
A kit for medical use, including. a) The recombinant bacterium according to any one of claims 1, 3 to 13, 15 or 17 to 21, capable of expressing at least one heterologous factor under the control of a chloride-inducible promoter of a prokaryotic system. ,
Including medical devices. 24. The pharmaceutical composition of claim 24 or the kit of claim 25 or claim 26, wherein the recombinant bacterium is frozen or dried in solution, preferably lyophilized or spray dried. Medical device. The pharmaceutical composition according to any one of claims 24 or 27 or any one of claims 25 or 27, wherein the recombinant bacterium is rearranged with a liquid containing chloride ions, preferably a culture medium. The kit or the medical device according to any one of claims 26 or 27. One of claims 25, 27, or 28, wherein the kit is provided as a combination preparation, preferably for the treatment of chronic inflammatory wounds, for separate, sequential, or simultaneous use. The kit described in. 25, 27, or 298, wherein the kit is provided as a combinational preparation for separate, sequential, or simultaneous use in the treatment of tumors, preferably malignant tumors. The kit described. Use of a repreparation medium containing chloride ions for repreparing the recombinant bacterium according to any one of claims 1, 3 to 13, 15 or 17 to 16.

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